JPH0568560A - Gene of bovin myogenic factor - Google Patents

Abstract

PURPOSE: To provide a novel DNA that is useful for synthesis of bovine myogenic factor (bmyf) protein.

CONSTITUTION: This gene is a cloned DNA containing the nucleotide sequence encoding bmyf active protein. The sequence-exemplified cDNA is isolated by screening the sequences homologous to myoD.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to cloning, expression, expression products, regulation and genetic engineering of genes involved in differentiation of progenitor cells into muscle cells (myogenesis).

[0002]

BACKGROUND OF THE INVENTION The present application is a priority claim application based on a part continuation application of a co-pending US patent application filed on December 7, 1990 (application number not yet attached). Is. The U.S. patent application filed on December 7, 1990 is a partial continuation application of U.S. Patent Application No. 454,080, which was filed on Dec. 21, 1989, and which belongs to the affiliation.

(Myogenesis at the Cellular Level) Myogenesis can be divided into three general stages (edited by Emerson, C. et al., 1986, Molecular Biology of Muscle Developm).
ent , Alan R. Liss, Inc. NY). First, mesodermal cells are transformed into proliferating myoblasts. Second, myoblasts fuse to form multinucleated myotubes. Muscle-specific genes are activated in this myotube. In the first decision stage, the myogenic lineage is taken over by the cell and all its progeny. In the second terminal differentiation stage,
Myoblasts form non-dividing myotubes that activate a set of muscle-specific genes. In the subsequent third stage, a complex series of regulatory events accompanies maturation in which various muscle protein isoforms are formed. Since proliferative myoblasts themselves can be identified as subclones that take over the activation of the embryonic heavy chain myosin gene that is either fast-twitch or slow-twitch,
Seems to be different.

Although the step leading to the origin of mesodermal cells was not involved in this pathway, many details of the cellular pathway to skeletal muscle have been studied in the early embryo. Skeletal muscle is derived from the middle layer, ie, the mesoderm layer, out of the three germ cell layers (the surface layer, the inner layer, and the mesoderm layer) that are organisms. Mesoderm is a pluripotent tissue that differentiates into many cell types except for skeletal muscle, including: That is, the cell types include bone, cartilage, and myocardium; eyes, teeth, kidneys and parts of the gonads; and a large number of cells in the hematopoietic pathway (red blood cells, lymphocytes, macrophages, etc.). The mesoderm is also identified by where it is formed in various regions of the early embryo (dorsal, head, lateral), and it is the dorsal region that produces morphologically visible somites and sarcomerees. The germ layer. Skeletal muscle is differentiated from these somites and sarcomere. Thus, the decision involves the conversion of dorsal mesoderm cells to proliferative myoblasts, resulting in the inheritance of myogenic lineages by mesoderm cells and their progeny.

An industrial concern for genes that regulate myogenesis is their use in promoting recovery of muscle damage. Muscle contains satellite cells, which are dormant myoblasts. The ability to distribute satellite cells to the site of injury and differentiate into myotubes may lead to the development of new medicines that increase the rate of healing of injuries, especially muscle trauma. Other industrial concerns are focused on livestock. This is because, in this field, the use of a myogenic gene can improve productivity with high efficiency and reduce the use of feed additives. At present, additives such as estrogens and antibiotics are widely used to achieve maximal muscle gain and weight gain. However, these additives may remain in human food and cause the growth of microorganisms resistant to antibiotics,
Its use is often criticized. Myogenic genes can also act on livestock before and after birth to reduce fat accumulation and increase myogenesis. As a result, lean meat (eg, low in fat and cholesterol)
Or healthier human food. Many substances developed from myogenic genes have commercial diagnostic uses in veterinary and human medicine.

Recently, mouse C3H10T1 responds to an effective inhibitor of DNA methylation (5-azacytidine).
Significant advances have been made in the isolation of genes involved in establishing muscle cell lineages by exploiting the unusual properties of cells. After treatment with this agent, colonies containing polynuclear myotubes form in few survivors (Constantides,
PG et al. (1977) Nature 261 : 364; Taylor, SM and J.
ones, PA (1979) Cell 17 : 771; Jones, PA and T
aylor, JM (1980) Cell 20 : 85). By isolating unfused myoblasts, stably growing and inducing
Myotubes in which muscle-specific genes are activated (eg, myosin H chain, myosin L chain (lemb, 1, 2, 3),
Differentiate into α-actin, α, β-tropomycin, and tropomycin C and T) (Konieczny and Eme)
rson (1984) Cell 38 : 791-800).

These observations indicate a simple “master gene” hypothesis (Konieczny, S. in Emerson et al., Supra).
(See F. et al.). This hypothesis is based in part on the fact that genes that are slightly methylated for cytosine residues are normally actively transcribed, whereas genes that are highly methylated are transcriptionally inactive. .. According to this model, a single gene (eg,
Myogenic determination gene) is activated by demethylation reaction,
10T1 / 2 cells are transformed into proliferating myoblasts that can differentiate into myotubes. When DNA derived from myoblasts (quail and mouse) and not derived from non-muscle cells is introduced into 10T1 / 2 cells by transfection,
This model was confirmed because it was shown that these 10T1 / 2 cells could be converted to myoblasts (Konieczny, SF et al. In Emerson et al., Supra; Lassar, A.
B. et al. (1986) Cell 47 : 649-656). Also, the unmethylated human DNA, were able to induce 10T1 / 2 cells myogenic lineage (Pinney, DF et al. (1988) Cell 53: 78
1). If DNA from widely distributed species (ranging from birds to humans) functioned similarly in the transfection assay, gene function was significantly conserved.

(MyoD gene lineage) As an analysis method,
Using transfection of 10T1 / 2 cells with DNA, myoD (Davis, RL et al. (1987) Cell 51 : 98
Two different genes named 7) and myd (Pinney, DF et al., 1988) were first identified. These genes transform 10T1 / 2 cells into myoblasts capable of forming myotubes. cDNA for myoD1 is 10
It has been cloned from a subtracted cDNA library from T1 / 2 myoblasts and sequenced (Davis et al., 1987). Its myogenic activity was analyzed as a chimeric gene using a retroviral promoter. myd has not yet been cloned. According to the hybridization standard in Southern blot, m
yoD and myd are not related to each other (Pinney
Et al., 1988). Biochemistry research shows that myoD is 30-6
It has been shown to be a nuclear phosphoprotein that converts with a half-life of 0 minutes (Tapscott, SJ et al. (1988) Science, 242 :
405). The transcript is also unstable (Thayer, MJ et al. (19
89) Cell 58 : 241).

[0009]

SUMMARY OF THE INVENTION A novel gene involved in the regulation of myogenesis (in the present application, its expression product, bovine myogenic factor (bm
(named for yf)) was isolated as a cDNA and characterized.

The bmyf cDNA was, unexpectedly, isolated in a screen for sequences homologous to myoD. The present invention provides a bmyf coding sequence, bmy
fcDNA, bmyf genomic DNA, promoter of bmyf gene, bmyf protein, polyclonal and monoclonal antibodies against bmyf protein,
Provide a chimeric gene construct of a bmyf promoter and a heterologous gene to be regulated so that bmyf is regulated in vivo, and a bmyf coding region that expresses bmyf under conditions other than those that normally induce bmyf It is a thing. The cDNA encoding bmyf has been cloned into bmyf in various art-recognized expression systems.
It is useful for large-scale synthesis of f protein. This cDNA is also useful for constructs that improve muscle maintenance, or for genetic modifications that increase weight gain and meat production efficiency. For example, a transfected cell line containing the above chimeric gene construct can be used to screen for compounds that can affect myogenesis.
The bmyf genomic gene is useful as well, but is particularly useful for gene replacement and gene therapy applications. bmyf
Promoters are particularly useful as regulators of reporter genes that are desired to be activated or repressed in contact with other myogenic genes. This promoter is also useful for analytical systems that measure the effects of compounds as potential regulators of myogenesis. Screening for compounds that affect myogenesis as therapeutic agents or regulators of muscle development can be performed using the construct in which the bmyf promoter and an appropriate reporter gene are linked. These constructs are DNA
It can be introduced into various cell lines by transformation. Transformed cells contain bmyf protein or b
It is a model for screening compounds that affect the activity of the myf promoter. Although the bmyf protein itself acts as a pharmacological agent, it is more likely to be used as a model for the production of smaller molecules with similar effects. In addition, bmyf protein is
It is useful as an antigen for producing and selecting polyclonal and monoclonal antibodies against bmyf. The antibody itself is useful as a diagnostic tool to analyze bmyf in cells under normal and pathological conditions. The effect of treatment with various drugs on the bmyf promoter or protein can be monitored by an antibody against bmyf. Monoclonal antibodies against various bmyf anti-determinants are useful in mapping the functional domains of the bmyf protein. The chimeric gene construct of the bmyf promoter and the heterologous coding sequence provides a test system for determining the effect of various compounds on normal and pathological bmyf expression as well as the transfected cell lines. Chimeric gene constructs with an exogenous promoter and a coding sequence for bmyf (cDNA or genomic sequence) are useful for therapeutic replacement in the presence of genetic conditions characterized by aberrant expression, or for example in feed It is useful to provide higher expression levels and increase meat production under the control of external stimulants such as additives. The above is illustrative of the applications in which the products of the present invention should be used and is not a complete listing of the applications.

Those skilled in the art can use the well-established and characterized methods known in the art for bmyf cD
Based on the availability and characterization of NA, various products of the invention can be isolated and used to characterize.
For example, bmyf cDNA serves as a probe to isolate genomic bmyf DNA from a bovine genomic library. Many expression systems or baculovirus eukaryotic expression systems that express bmyf cDNA to produce bmyf protein, including fusion proteins to bacterial trpE gene proteins, are known in the art. By isolating the bmyf genomic clone, the elements that control the transcription of the bmyf gene, including the promoter of the bmyf gene, can be identified. After sequencing by well known methods, this promoter can be used to ligate with a heterologous coding sequence to construct a chimera. With the disclosed cDNA coding sequences, one of skill in the art can also construct chimeric genes in which exogenous or heterologous promoters, such as those of retroviral or muscle-specific genes, control the expression of the bmyf coding sequences. If the whole bmyf protein or various subregions provided by peptide synthesis are used as antigens,
Well-known methods for producing polyclonal or monoclonal antibodies are available. Accordingly, one of ordinary skill in the art, given the data, information, and teachings disclosed herein, can readily utilize various products of the present invention. Alternatively, the activity of the bmyf promoter or polypeptide may be increased or decreased, or the bmyf polypeptide or m
Well-known techniques for increasing or decreasing RNA stability can be used to introduce conservative nucleotide sequence changes (changes that do not result in a modified amino acid sequence) into a DNA sequence.

The present invention relates to bmyf protein or bmyf
Provided is a cloned DNA (eg, cDNA, genomic DNA) containing a nucleotide sequence encoding an active protein. The present invention also provides DNA containing the promoter of the bmyf gene. Furthermore, the present invention provides such cloned DNA or cultured cells transformed with such DNA, purified protein having an amino acid sequence containing the amino acid sequence of bmyf active protein, polyclonal antibody or monoclonal antibody specific to bmyf protein,
And a proteinaceous composition comprising these polyclonal or monoclonal antibodies.

[0013]

DETAILED DESCRIPTION OF THE INVENTION Generally, the terms used in this application are standard terms understood by those of ordinary skill in the art of molecular biology, genetics, and cloning. Some terms are further defined for greater clarity. Scientific journals in the above fields (Nature, Cell, Proceedings of the Nationa
Standard abbreviations accepted by the Academy of Science USA, Science, etc.) are used.

The term myogenesis is a term used in the multi-step process of cell differentiation in which non-muscle stem cells are converted into muscle cells. Myogenesis is the production of myoblasts, mRNA or protein of muscle-specific genes (H and L chains of myosin, α-actin, α-tropomyosin and β-tropomyosin, and troponin C and troponin T).
Expression, myotube formation, fusion cell generation, and muscle-specific promoter activity are recognized by any of several criteria. The currently available analytical method for the early stages of myogenesis uses the publicly available embryonic mouse fibroblast cell line C3H10T1 / 2. C3H10T1 / 2 cells undergo myogenic conversion by transfection with an expressible myogenic gene such as MyoD or by treatment with 5-azacytisine. In the present invention, C3H1 by bmyf cDNA in an expression vector such as pEMSV-bmyf.
It is shown that transient transfection of 0T1 / 2 cells elicits a myogenic response in C3H10T1 / 2 cells.

The term promoter is used to indicate a gene region that has the function of controlling the transcription of a gene. The promoter is typically 5'of the coding region
It is located near the end (the region encoding the amino-terminal portion of the encoded protein). As is known in the art, the location of the promoter is based on the genomic DNA
Sequence analysis and other approved methods (S1 mapping method, DNAseI footprinting method, gel delay analysis method, and transient transfection method with promoter and reporter gene). Promoters include sequences that vary in size and have more than one regulatory function (eg, regulatory functions with responses to inducers, repressors, and activators and tissue specificity, etc.).

The bmyf active promoter is a variant of the nucleotide sequence of the bmyf promoter sequence,
It has at least 80% homology to the yf promoter sequence and has substantially the same function as the bmyf promoter. The bmyf promoter controls the transcription of the bmyf gene, has an RNA polymerase binding site,
It contains a regulatory region that activates and / or represses the transcription of the yf gene. The regulatory region may be located 5 ′ to the structural gene, within the structural gene (including introns), and / or 3 ′ to the structural gene. As discussed in the Examples below, the regulatory region of a promoter and its function can be identified by methods well known in the art.

The term coding region is used to indicate the part of the gene that codes for a protein. cDNA
In, a single polypeptide chain is encoded by a continuous uninterrupted coding region. In genomic DNA (eg, the gene itself), its coding region is usually interrupted by one or more introns. These introns are non-coding intervening sequences present in the gene, but not in mRNA.
A coding region is under the control of this promoter if it controls the expression of the protein encoded by the coding region.

Genomic DNA is DNA that is directly cloned from the chromosomal DNA of an organism. The bmyf gene is a genomic D cloned from the chromosomal DNA of an organism.
Included in NA. Thus, genomic DNA is
Before transcription, shows the structure of a gene in the chromosome of an organism.

The term expression means that the presence of a gene in a cell or organism becomes detectable. Expression occurs whenever a phenotype attributable to the gene is observed. More directly stated, gene expression is the synthesis of a gene product by transcription into mRNA or translation of mRNA into protein.

The term chimeric gene is used to indicate an artificial gene constructed by joining together functional genetic elements obtained from different sources. For example, bmyf
A chimeric gene can be constructed by linking a promoter to the coding region of another gene. Such coding regions can be found in bmy in naturally occurring cells.
It is called the heterologous coding region because it is not regulated by the f promoter. If the heterologous coding region is expressed to provide a readily detectable product, the chimeric gene is used as a reporter gene to measure the activity of the promoter under experimental conditions. Another example of a chimeric gene is a gene constructed by joining an exogenous or heterologous promoter with the bmyf coding region. The exogenous promoter is
It is a promoter that does not regulate the bmyf gene in naturally occurring cells.

The bmyf protein is an expression product of the bmyf gene. The deduced amino acid sequence of the bmyf protein is shown in SEQ ID NO: 3.

The bmyf active protein is an amino acid sequence variant of the bmyf protein having at least about 80% sequence identity with the bmyf protein and having substantially the same activity. Therefore, the bmyf protein is
It is known to function as a regulatory protein that plays a fundamental role in controlling myogenesis in mammalian cells. Further studies will confirm its function more quantitatively and clarify the molecular mechanism of its activity. It is understood that many amino acid sequence variants are possible that have substantially the same activity. Such variants include, but are not limited to, conservative amino acid substitutions as well as substitutions and deletions in non-critical regions of the protein. Such variants are called bmyf active proteins, as long as they have substantially the same activity as bmyf protein itself (distinguishable from other proteins of myogenesis and quantitatively similar), Included within the scope of the invention.

The methods used herein are either specifically cited or are well known and are available in several publications which summarize the methods. Publications include, for example, Maniatis et al., Molecular Cloning, a Labor.
atory Manual , Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York (1982); Genetic Engineerin
g , Plenum Press, Ner York (1979); Weir (1989),
Of the four volumes, Handbook of Experimental Immunology , Fourth Edition, Blackwell Scientific Publications, Oxford;
And many volumes in Methods in Enzymology , Acad
There are emic Press Press and New York.

Various cell lines and plasmid vectors have been disclosed. All are publicly available. bmyf
Cells or tissues that serve as a source of cDNA or bmyf genes are readily available. The plasmids, cloning vehicles, and cells used for clonal transfer and expression were chosen for ease of manipulation. Substitutes can be used at any stage, paying attention to the desired properties, according to criteria and properties known to those skilled in the relevant art.

Examples of the present invention will be described below. In these examples, the bmyf cDNA of the invention is
And the cloning and characterization of other products.

Example 1 Construction of cDNA Library and
Gosp except that selection by screening cytochalasin was omitted
Bovine myogenic cells were isolated from fetal thigh muscle as described by odarowicz et al. (1976) J. Cell. Biol. 70 : 395-405. Cells were treated with 4M guanidine thiocyanate, 0.5% (v
/ v) Sarkosyl, 0.025M EDTA, and 0.1M β-mercaptoethanol were used for dissolution. Chirgwin et al. (1979) Bioch
emistry 18 : 5294-5299; Freeman et al. (1983) Proc.Natl.Ac
Cs as described in ad.Sci.USA 80: 4094-4098.
RNA was isolated by precipitation of Cl. RNA pellet for 10
mM Tris ・ HCl (pH7.6), 1 mM EDTA, 5% (v / v) Sarkosyl,
And dissolved in 5% (v / v) phenol and then extracted with phenol: chloroform: isoamyl alcohol (50: 49: 1). After ethanol precipitation, RNA was dissolved in deionized water and stored at -70 ° C. Poly A ⁺ RNA was isolated by chromatography using oligo (dT) -cellulose,
Used to synthesize first-strand cDNA with AMV reverse transcriptase and oligo (dT) as primer (Berger and Kimmerl (1987) Meth. Enzymol. 152). Second-strand synthesis was performed using DNA polymerase I under storage of RNase H (Gubler (1987) Meth. Enzymol. 152: 330-335). CL
Chromatography with -4B Sepharose
CDNAs with an average length of 500bp or more were isolated, methylated, and Ec
It was ligated to the oRI linker at the blunt end. The cDNA bound to the linker was cleaved with EcoRI, fractionated according to size, and
It was cloned into the EcoRI site of gt10. Commercially available extract (Pack
^{ageneR TM system; Promega, Medison,} WI) using, after the recombinant phage DNA was packaged, to amplify the library.

A phage library containing about 3 × 10 ⁴ recombinants was used as a template for pVZC11b plasmid DN.
Using a ³² P-labeled 274 bp fragment of mouse MyoD1 cDNA amplified by polymerase chain reaction (PCR) using A,
Thorough screening (Sakai et al. (1985) Science 23
0: 1350-1354; Mullis and Faloona (1987) Meth, Enzymol,
155: 335-350). A pair of degenerate 1's starting at nt339 and 612 of the sense and antisense cDNA strands, respectively.
An 8-mer primer was synthesized and used to amplify this 274 bp fragment. This fragment spans the cys / his region, the basic region, and the myc homology region of MyoD1 and is randomized to oligonucleotide priming (Feinberg and Vogelstein (1983) A
nal.Biochem. 132: 6-13), high specific activity (10 cpm / μg or more) was labeled with ³² P- (α) dATP. For screening libraries, standard methods (Car
plaques were bound to nylon filters (Micron Separations, Inc) using lock (1986) Focus 8: 6-8).
Heat these filters at 70 ° C for 60-90 minutes and
× SSC (0.9M NaCl, 0.09M sodium citrate, pH 7.0),
0.05% (w / v) SDS, 0.25% (w / v) Blotto, 1 x Denhardt's solution (0.02% (w / v) Ficoll, 0.02% (w / v) Polyvinylpyrrolidone, 0.02% (w / v) BSA), 0.05% (w / v) sodium pyrophosphate, and 5 mg / ml denatured salmon sperm DNA, 50
Prehybridization was performed at 6 ° C for 6 to 12 hours. Hybridization consisted of 6 x SSC, 0.5% (w / v) SDS, and 0.
It was carried out at 50 ° C. for 24 hours in a buffer containing 05% (w / v) sodium pyrophosphate using a denaturing probe at 10 cpm / ml. Next, the filter is 2 × SSC, 0.1% (w / v)
Twice in SDS at room temperature, 5 in 1 × SSC, 0.1% (w / v) SDS
5 times in 0.5 x SSC, 0.1% (w / v) SDS at 4 ° C
It was washed once at 4 ° C. Each wash lasted 15 to 30 minutes. After exposure of the filters to Kodak X-0MAT film, positive recombinants were purified by serial dilution and rescreened under the conditions described above (Carlock et al., 1986).

Example 2 Characterization of cDNA clones Recombinant phage DNA was cleaved with BamHI and electrophoresed on a 0.7% agarose gel, 1M ammonium acetate and 0.02N NaOH (Rigaud et al. (1987) Nucl. Acids Res. 15 : 8
57) in Biotrans nylon filter (ICN Biomedicals
Inc, Costa Mesa, CA). The filters were UV-irradiated, heated at 70 ° C. and hybridized to the 274 bp probe, essentially as in the library screen above. Recombinant insert, pGEMTM-3zf (-)
(Promega) was subcloned into the EcoRI site to create a restriction endonuclease map. Various subfragments are M13
Subcloned in and sequenced on both strands by the dideoxy method of Sanger et al. (1987). The restricted part is
Especially when it is missing in the 3'untranslated region of bmyf,
A 17mer was synthesized to complete the sequencing.

The complete nucleotide sequence of the bmyf insert is shown in SEQ ID NO: 2. This nucleotide sequence is
It contains 1,889 bases (excluding the short poly A portion) and contains a long open reading claim of 765 nucleotides beginning at the potential ATG start site at nucleotide 69. The deduced amino acid is a protein derived from the longest open reading frame of human myf-5 cDNA (Braun et al. (198
9) It can encode a protein of 255 amino acids with the same size as EMBO J.8: 701-709). bmyf and human myf have extensive homologous regions, especially in their coding regions. 96% identical at the amino acid level and 92% identical at the nucleotide sequence level. Both cDNAs contain a short segment (68 in bmyf) upstream of each potential ATG start codon.
Nucleotides, 42 nucleotides in human myf-5).
These regions are also highly related, exhibiting 82% sequence conservation. Although it is unclear whether the first nucleotide is the actual 5'transcriptional start site of bmyf mRNA, a separately cloned smaller bovine cDNA (clone 7) contains the same nucleotide containing the first nucleotide. It has a 5'side sequence. This suggests that bmyf is indeed a full-length cDNA.

The main difference between bmyf and human myf-5 is that bmyf
Has a 3 ′ untranslated region that is longer by several hundred nucleotides. From the stop codon, AA at nucleotide 1407 in human myf-5 and at nucleotide 1415 in bmyf
2 up to the TAAA polyadenylation signal
The two cDNAs are very related and 81% of the sequences are similar. Human myf-5 terminates within a short distance of this polyadenylation signal, while bmyf extends an additional 475 nucleotides, the second at nucleotide 1872.
It terminates near the polyadenylation signal of. Probably
Human myf-5 cDNA is derived from a transcript that terminates at upstream sites, whereas bmyf is believed to be derived from a transcript that terminates at downstream sites. As described below, RNA from fetal skeletal muscle contains a 1.5 kb bmyf transcript that is believed to result from adenylation at the AATAAA site at nucleotide 1415.

Example 3 Northern Hybridization
It was excised various tissues from emission analysis 20cm fetal bovine and frozen in liquid nitrogen. Skeletal muscle was taken from the hind and forelimbs. To prepare RNA, frozen tissue was ground into a fine powder and 5M guanine thiocyanate, 10 mM EDTA, 50 mM Tris-HCl (pH 7.
5) and 8% (v / v) β-mercaptoethanol. RNA was precipitated directly from the lysate using 4M LtCl as described above (Cathala et al. (1983) DNA 2: 329-33.
Five). After solubilization and extraction with phenol, RNA was precipitated with ethanol to give LiCl pellets or an aqueous solution.
Stored at 70 ° C (Cathala et al., 1983). Poly A ⁺ RNA,
Isolated as described above, electrophoresed on 1.1% agarose formaldehyde and transferred to Biotrans nylon membrane in 20xSSC. The filter was irradiated with UV, heated at 70 ° C. for 60 to 90 minutes, and hybridized with ³² P-labeled cDNA as follows. 0.5M sodium phosphate (pH7.2), 7% SDS, 1
Prehybridize the filters in% BSA and 1 mM EDTA at 65 ° C for 4 hours, then use base-denatured probe (10 ⁶ cpm / ml or more) under the same conditions for 18-2.
Hybridized for 4 hours (Mahmoudi and Lin (198
9) Biotechniques 7 : 331-333). Use a 40 mM sodium phosphate buffer (pH 7.2), 5% (w / v) BSA, 5% (w
/ v) SDS and 1 mM EDTA at 65 ℃ for 30 minutes each, 2
Washed twice, 40 mM sodium phosphate (pH 7.2), 1 mM EDTA,
And washed twice in 1% (w / v) SDS at 65 ° C (M
ahmoudi and Lin, 1989). The probe was gel purified before use (Dretzen et al. (1981) Anal. Biochgem 112 : 29.
5-298).

When skeletal muscle-derived poly A ⁺ RNA was analyzed by Northern blot using the full-length bmyf cDNA as a probe, it was detected that three kinds of RNA migrated to 1.5 kb, 2 kb, and 3 kb. The 1.5 kb band is most abundant and is present at levels 2-3 times higher than the high molecular weight species. The presence of three different bands in this Northern blot indicates that bovine
It shows that transcripts from the myf gene are processed separately. For example, the 1.5 kb RNA band is equivalent to the transcript that produces human myf-5 cDNA, and may be lacking the region downstream of the proximal polyadenylation signal of bmyf at nucleotide 1415. To investigate this, Northern blots of poly A ⁺ RNA from bovine skeletal muscle were derived from the distal end of bmyf at nucleotide 14
354 bp, 3'to the polyadenylation signal at 15
It was probed with the AvaII / EcoRI fragment. The distal 354bp fragment is 2
Hybridized with kb and 3 kb bands, but 1.5 kb
Substantially did not hybridize with RNA. This indicates that this species lacks the same portion of the missing 3'sequence of the human myf-5 cDNA.

To examine whether bmyf is expressed in a tissue-specific manner, poly A ⁺ RNA derived from several fetal bovine tissues was examined.
Was subjected to Northern blot analysis and probed with the full-length bmyf cDNA. Only RNA from skeletal muscle gave a signal. The actual level of bmyf transcript present in skeletal muscle was low, compared to the signal produced by known amounts of bmyf RNA synthesized from the pG EMTM vector containing the bmyf insert, 0.001% to 0.0001% of total message. It is estimated to be. When RNA from the mouse azamyogenic cell line was subjected to Northern blots at the level of stringency used above, little or no code was detected.

Example 4 Cell culture and DNA transfer
CHT obtained from Ekushon American Type Culture Collection
1/2 (clone 8), 10% heat inactive FCS, 100 μg / m
l DMEM (Cellogo ^TM , Mdiatech, W
(A. Washington, DC). A stable azamyogenic cell line was obtained by treating C3H10T1 / 2 cells with 3 mM 5-azacytidine for 24 hours and serially subcloning them (Konieczny and Emerson, 1984). When plated at low density, over 90% of the colonies derived from the 91a and 56a azamyogenic subclones were positively stained for a monoclonal antibody against the heavy chain of skeletal myosin (MF-20, see below). A large number of multinucleated fused cells were formed.

DNA transfection was carried out using pEMSV-scrib
This was done using the Moloney sarcoma virus LTR of e and a cDNA inserted at the EcoRI site between the SV40α polyadenylation signal (Harland and Weintraub (1985) J. Cell.
Biol. 101 : 1094-1099; Davis et al., 1987). Full length bmy
The f cDNA was inserted in both sense and antisense orientations. In transient transfection (Wright et al. (1989) Cell 56 : 607-617), C3H10T1 / 2 cells were
1 × 10 ⁵ cells were plated per 10 cm dish and grown in DMEM containing 10% heat-inactivated fetal bovine serum for 2 days. Replaced 3 hours before transfection and described (Gorman
(1985) DNA Cloning VolII, IRL Press, Oxford UK, p
p.143-190), calcium phosphate precipitates (7.5
Plasmid DNA was added to μg / dish). The cells were incubated for 24 hours and then phosphate buffered saline (137 mM NaCl,
Wash with 2.7mM KCl, 4.3mM Na HPO, and 1.4mM KH PO) and differentiate with differentiation medium (D containing 2% (V / V) heat-inactivated horse serum)
They were maintained in MEM) or grown in normal medium for 2 days before incubation in differentiation medium. Three
After a day, the cells were treated with 70% (V / V) ethanol, 3.7% (V / V)
Immobilized in formaldehyde and 5% (V / V) glacial acetic acid, and then reacted with a monoclonal antibody against the heavy chain of skeletal muscle myosin (MF-20, Bader et al., 1982) and then bound to alkaline phosphatase. Goat anti mouse Ig
G was made to react. For immunostaining, NBT and BCIP staining reagents
(Promega) and following the supplier's instructions.
Micrographs were taken with a Kodak Panatomic-X film using the Z
Photographed with an eiss inverted microscope.

A transient assay using the same vector
Bovine myf cDNA was transferred to C3H10T by transfection.
It shows that 1/2 cells can be converted to myoblasts. b
With the above expression vector having myf in the sense direction, C3H10T1 / 2
Cells were transfected, induced to differentiate for 3 days, then fixed and even reacted with a monoclonal antibody against the heavy chain of skeletal muscle myosin. Cells were transfected with the same vector with bmyf in the antisense orientation or mouse Myo D1 in the sense orientation (as negative or positive controls, respectively). Many cells were positive for antibodies in cultures transfected with the bovine myf expression plasmid in the antisense direction that reacted positively with the myosin antibody probe by transfection with the bmyf expression plasmid in the sense direction. There wasn't. Cells transformed to the muscle myosin phenotype with the bovine myf expression vector were present in both experiments at frequencies of 10 ^-3 and 10 ^-4 . This frequency is several times greater than the spontaneous activation level in 10T1 / 2 cells (Lassar et al.,
1986). It was observed that both mononuclear cells and polynuclear cells stained positive for heavy chain myosin.

Example 5 bmyf gene and its proximal progeny
By comparing the restriction enzyme map of the motor structure bmyf cDNA with the fetal bovine skeleton genomic DNA, it was presumed that an intron was present in the bmyf gene (Fig. 1). To determine the exact location of the intron in the bmyf gene, the bmyf genomic fragment was amplified by polymerase chain reaction (PCR) using a set of sense and antisense bmyf cDAN primers (US Pat. No. 4,683,202). No.). The amplified genomic fragment containing the larger intron (Intron A in Figure 1) was subcloned and the exact location of the intron was determined by partial sequencing using the dideoxy chain termination method. This fragment was predicted to contain an approximately 1 kb intron between cDNA residues 569 and 570. The restriction enzyme map of genomic DNA (Fig. 1) shows EcoR in intron A.
The I and Xba I sites are indicated. The second amplified fragment contained a smaller intron of approximately 300 nucleotides (Fig. 1
Of the intron B).

The overall structure of the bmyf gene is mouse myf-5.
Is very similar to the herculin gene adjacent to (J.
H. Miner and B. Wold, PNAS USA 87 : 1089-1093
(1990)). This similarity is consistent with the idea that the bmyf and herculin genes arise from sequence divergence from older gene duplications in ancestral genes.

To isolate the proximal bmyf promoter,
"Inverse" PCR was used to increase the 5'region of the cDNA. First, fetal bovine skeletal muscle genomic DNA was digested with baI and EcoRI. As mentioned above, the restriction sites for XbaI and EcoRI were determined to be in intron A. next,
By re-annealing this cleavage product under conditions suitable for ligation of circular molecules, a 930 bp circular fragment consisting of an XbaI fragment extending from −218 to +145 of intron A for cDNA was obtained.
A DNA molecule was formed. With two primers starting at +466 for sense and +218 for antisense, the region between the two primers containing the XbaI site was increased by PCR. This region contained the 5'region of the cDNA.

The 281 bp XbaI / PstI fragment derived from the 930 bp fragment was subcloned and sequenced by the dideoxy chain termination method. The sequence revealed that this fragment contained nucleotides 1-67 of the bmyf cDNA, which
A portion of the 930 bp fragment up to the −218 XbaI site is actually a cDNA
It was found to be present on the 5'side of the mouse and represents the proximal promoter.

Example 5.1 Bmyf fragment derived from bovine genomic DNA
Isolation of Genes To isolate fragments containing genomic bmyf DNA inserts, a commercially available bovine genomic library was screened as follows. (1) a fragment containing the bmyf gene is amplified by PCR using the intron A primer, and (2) above.
The amplified fragment of (1) was screened using the full-length bmyf cDNA as a probe.

The 20,000 recombinants of the Stratagene 1 DASH ^™ II genomic library contain a partially degraded fragment of Sau 3A of bovine genomic DNA. This was seeded on each of 10 large plates. Plate culture lysates were assayed by amplification by PCR with the Intron A primer in the presence of the bmyf gene. Four plates were positive and one was thoroughly screened with the full length bmyf cDNA probe.

ΛDII bmyf1 which is one of the positive plaques
Contains an insert of approximately 13 kb, and its partial restriction map (Fig. 1) shows that it contains the bmyf gene. The bmyf gene (about 3 kb) is flanked at its 5'end by a fragment of about 4 kb, and also at its 3'end by about 7 kb.
flanked by kb fragments. This 13 kb clone was confirmed to contain the bmyf gene based on the fact that the 217 nucleotides of the first exon show complete sequence homology with the sequence of the bmyf cDNA.

In order to determine the sequence of the bmyf coding sequence and the promoter portion, various portions of 13 kb were digested with pGEM.
Subcloned into the EcoRI polylinker site of ^TM- 3zf and -zt vectors (Promega). Isolate single-stranded DNA and accelerate the synthetic reaction using the dideoxy chain termination method 20
Sequencing was performed using mer oligonucleotides. The complex of the cDNA with the available genomic sequence is shown as the 5'to 3'sequence in SEQ ID NOS: 4-12 of the Sequence Listing. cD
The NA nucleotide sequence and the corresponding bmyf amino acid sequence are shown in SEQ ID NO: 1. The cDNA nucleotide sequence is shown in SEQ ID NO: 2. The bmyf amino acid sequence is shown in SEQ ID NO: 3. In SEQ ID NOS: 1 and 2, the coding region of the cDNA sequence is shown by triplet codons, and the non-coding region of the cDNA is shown as a nucleotide sequence in blocks of 10.

SEQ ID NO: 4 begins near (but not including) the BamHI site approximately -4500 nucleotides upstream from position +1 of the bmyf cDNA. The length of the sequence of SEQ ID NO: 4 is
It is 385 nucleotides.

SEQ ID NO: 5 begins at the Sac I site (GAGCTC) approximately -2300 nucleotides upstream from position +1 of the bmyf cDNA. The length of the sequence SEQ ID NO: 5 is 815 nucleotides. Between SEQ ID NO: 5 and SEQ ID NO: 6 is an approximately 440 bp unsequenced segment.

SEQ ID NO: 6 is the proximal promoter sequence starting at -946 nucleotides upstream from position +1 of bmyf cDNA. This sequence attaches directly to position +1 of the bmyf cDNA. Based on the sequence information of the promoter region, this promoter is rich in GC and has three TATA motifs (cDNA +1
Present at −158, −114, and −60 nucleotides upstream of position). This motif is the RNA polymerase
TF, a binding transcription factor required for II-mediated transcription
Can serve as a site for IID. 5'determining the TATA box that is thought to be used as the transcription start site
Footprinting of the area will be described later.

SEQ ID NO: 7 is bmyf cDN starting from position +1
It is the part of A and continues up to +569 of the cDNA, from there the genome
DNA intron A begins. The coding region of the cDNA sequence is indicated by the triplet codon. The non-coding region of the cDNA is shown as a nucleotide sequence in blocks of 10.

SEQ ID NO: 8 is a sequence encoding a part of intron A. Intron A is approximately 1000 bp and is not a complete sequence. SEQ ID NO: 8 is intron A
Is 361 bp at the 5'end. The region following SEQ ID NO: 8 is approximately 4
This is an unsequenced region of 00 bp.

SEQ ID NO: 9 is 400b which has not been sequenced
It is 258 bp of intron A downstream of the p region.

SEQ ID NO: 10 is a sequence directly following SEQ ID NO: 9 and starts from the coding region corresponding to the +570 position of bmyf cDNA.

SEQ ID NO: 11 is the entire sequence of intron B. Intron B is +644 and +65 of bmyf cDNA
Located in the genomic DNA in the part between 5. Intron B is
It is 393 bp long.

SEQ ID NO: 12 is the sequence of exon 3. Exon 3 begins at position +646 in the cDNA. This is 1
It is 245 bp long and extends through the poly A attachment site.

Example 5.2. Transcription start site of bmyf gene
Mapping As mentioned above, the proximal promoter region contains three TATA elements from which transcription can be initiated by RNA polymerase II. RNAase protection experiments were performed to identify the most likely starting site.

An antisense RNA probe labeled with ³² P (extending from PstI at +64 of cDNA to XbaI at −218 upstream) was annealed to total RNA from fetal bovine skeletal muscle (18 cm fetus). It was The annealed mixture was cut with RNAse A and TI. The protected double-stranded fragment is then denatured and then separated on a sequencing gel,
Its size was measured by comparison with a series of dideoxy sequencing reactions over the same region. According to this method, the transcription initiation site is in the vicinity of about -185 nucleotides upstream of PstI at the + 64th position of cDNA, or cDN
It was determined to be in the TATA motif in the vicinity of -114 nucleotides upstream from the +1 position of A.

Judging by the intensity of the probe signal, the early fetus is more likely than the later fetus (eg, 18-
The 42 cm fetus) promoter is considered to be highly useful.

Example 6 Chimeric Constructs Subclones of the bmyf gene containing only the Bmyf coding region or other desired regions of the bmyf gene can be constructed. For example, the sequence information provided herein identifies suitable restriction enzyme sites that can be used to construct and clone the desired segment. Other isolation techniques that do not require sequence information for the desired fragment may also be utilized. It especially includes PCR, which is described in US Pat.
No. 4,683,202. Synthetic primers can be constructed that have a sequence that groups together the desired segments, which allows the polymerase chain reaction, such as a portion of the bmyf promoter, bm
Any desired specific segment of the yf gene can be amplified. In addition, the primer sequences can be constructed to induce restriction sites with the desired specificity flanking the amplified segment, as is well known to those of skill in the art.

A chimeric gene construct of a segment of the bmfy promoter linked to a heterologous gene segment, or
It is possible to create a construct for an exogenous promoter that binds to the bmyf coding region, as will be shown later.

In addition, other parts of the bmyf gene can likewise be subcloned and linked into the chimeric functional gene. In this way, specific functional domains of the bmyf gene can be linked to other genetic constructs, or. It can be deleted from the bmyf gene to give rise to a different function (add or remove function associated with the subcloned domain).

Example 6.1. Chimeric bmyf promoter / CA
The functional chimeric gene in which the T gene construct bmyf promoter and the heterologous gene are linked also regulates the transcription of the heterologous gene, just as bmyf itself is regulated. The model code gene is
For example, in the case of a reporter gene that encodes chloramphenicol acetyltransferase (CAT), the resulting constructs are readily available for stimulants and chemical compounds that affect bmyf expression by either stimulation or repression. It will provide an index that can be measured.

To create a rough map of the functional region of the bmyf promoter, two overlapping duplicates of different length
The bmyf promoter part was linked to the chloramphenicol acetyltransferase (CAT) construct gene. These constructs were then transfected into cell lines and cell CAT activity was measured using ¹⁴ C-chloramphenicol.

One construct contained the full length of the 5'flanking portion of the 13 kb fragment, extending from the unique BamHI site of -4.2 kb to the PstI site of +64. 4.2kb promoter / CAT to construct 4.2kb promoter / CAT gene
To construct the gene, the 4.2 kb fragment has blunt ends at the 5'BamHI site and the 3'PstI site. pCAT ^T
M has a blunt end at the HindIII site in the polylinker,
It is then digested with PstI to form a site suitable for directional cloning of the 4.2 kb promoter fragment.

The second construct contains a proximal promoter segment extending from the XbaI site at -218 to the PstI site at +64. 282 bp for the proximal promoter / CAT construct
The XbaI / PstI fragment of E. coli was created by ligating to the pCAT polylinker site between the XbaI and PstI sites.

Each of these constructs can be used to transiently transfect 10T1 / 2 cells with an expression vector such as pEMSV, as shown in Example 4 above. This transient transfection is
It is performed to determine whether the fragment extending from the 4.2 kb BamHI site to the -218XbaI site contains the functional region of the promoter.

Example 6.2. Bmyf promoter cis segme
Useful chimeric bmyf the identification of cement promoter / CAT genetic
Examples To identify the regulatory portion of the progeny bmyf promoter
A construct confirmed to contain a functional promoter region in 6.1. And a series of deletion mutants of this construct were transfected into a cell line and expressed. The series of deletion mutations consist of progressive deletions from the 5'end of the promoter towards the transcription start site.

First, a large number of cell lines were transiently transfected with the full-length promoter / CAT construct in order to find cell lines capable of expressing the CAT gene lacking the bmyf promoter. For example, the myf-5 gene is L6
And C2C12 cells (Wright, W. et al. (1989) Cell, 56 : 607-671; Braun, T. et al. (EMBO J
8: 3617-3615); Peterson, C. et al. (1990) Cell 62: 493.
-502), these are the optimal target cell lines for transfection of the bmyf promoter / CAT construct. Furthermore, even though certain cell lines are believed not to express the CAT gene that lacks the bmyf promoter, testing for deletions of such cell lines indicates that these regions function as silencers for the bmyf promoter, ie Will activate the gene only when is removed).

Next, the full-length bmyf promoter / CAT construct and its deleted derivative were transiently co-transfected with RSV lac Z into selected cell lines (Sambrook, J.
Et al. (1989) Molecular Cloning; A Laboratory Manual
(2nd edition); Webster, J. et al. (1989) EMBO J 8: 1441-4
6) The CAT activity was measured using ¹⁴ C-chloramphenicol. Β-galactosidase activity is measured as an internal control of transfection effects between cell lines (Webster, J. et al. (1989) EMBO J 8: 1441-46). Transfection is performed by the calcium phosphate method.
(Clark, T. et al. (1990) Nucleic Acids Res 18: 3147−
3153; Glover, DM (1985) DNA Cloning: A Practical
Approach II, 143-190).

When the localization of this important part is carried out by analysis for deletions, the cis element can also be identified. The promoter region thought to be required for promoter activity can be confirmed by gel shift in vitro assay using extracts from cell lines known to express the bmyf promoter / CAT construct.

The promoter fragment determined to contain the cis element is subjected to DNAse footprinting in order to accurately elucidate the protected portion. DNAse footprinting is done by preparing two batches of the promoter fragment in question. One of the two batches is a trans-acting regulatory element (Trans
-batch containing cell extract with -acting regulatory element) and vat without cell extract, each fragment having radiolabeled 3'or 5'ends. So that on average there will be one cut per fragment for this batch,
The DNAse I is allowed to react rapidly, and then each batch is subjected to gel digestion to identify areas not cleaved by DNAse I (areas protected by the prepared protein).

Example 6.3 Identification of enhancer region
Use chimeric bmyf promoter / CAT structure Tsukibutsu constructs of Examples 6.1 and 6.3, as can be identified even a fragment believed to contain bmyf enhancer sequences, it can also be further modified. These sequences, like introns, can be located in the 5'and 3'flanks. Enhancers typically exhibit cell type specificity. The pCAT vector can insert such an enhancer sequence. Several unique restriction sites are included downstream of the CAT gene.
These restriction sites include EcoRI and BamHI.
The resulting construct can be transiently transfected into 10T1 / 2 cells and other cell lines by methods described above or known to those of skill in the art, and bmyf
Protein production was measured to determine the cell type specificity of the enhancer fragment in question.

Example 6.4 Chimeric bmyf promoter / β
-Galactosidase gene construct Another heterologous reporter gene used is the β-galactosidase gene. The bmyf promoter / β-gal gene construct was constructed using pCH110 (Hall, C et al. (1983) J. Mol. App.
l. Genetics 2 : 101-109; available from Pharmacia LKB), and pSP72 (Prom cleaved with BamHI / HindIII) by excising the lacZ gene on a BamHI / HindII fragment.
It can be created by inserting the lacZ gene into ega) with orientation. 4.2kb BamHI / Pst I bm
The yf promoter fragment was then transformed into XhoI and Pvu in pSP72.
It can be inserted into the II site by ligation with blunt ends. pSP7
2 contains a bmyf fragment containing the muscle-specific enhancer (4.2 kb
There are 7 restriction sites that can insert (other than the 5'fragment), which are downstream of the site where the lacZ fragment is inserted. These 7 restriction sites are SmaI, KpnI, SacI, EcoR
I, ClaI, EcoRV, and BglII. The β-galactosidase gene is useful as a reporter gene because it exhibits a staining reaction associated with β-gal activity and because antibodies against β-gal are commercially available.

Example 6.5 Chimeric pMMTV-bmyf gene and
And the pMMTV-myoD gene construct , a chimeric gene having an exogenous promoter linked to the bmyf coding region, expresses bmyf under conditions in which the action of the promoter is normally shown. This type of chimeric gene has a sufficient amount of bm for isolation and further characterization.
The yf protein can be expressed.

For example, the EcoRI restriction fragment containing the complete bmyf cDNA and myoD coding region is a pΩ5 vector.
(Morganstern, J. & Land, H. (1990) Nucleic Acids Re
s. 18 : 1068). This vector has an enhancer-free mouse mammary tumor virus (MMTV) promoter that is 20-50 fold inducible by dexamethasone in mammalian cell culture. The bmyf and myoD coding regions are in the sense direction (pΩbmyf + and pΩmyoD +)
And in the antisense orientation (pΩmyfl ⁻ and pΩmyoD ⁻ ).

Example 7 Expression and purification of bmyf in a cell line
The bmyf protein produced can be obtained by combining the chimeric gene of the present invention with any one of known expression systems. For example, by linking the coding portion of the bmyf gene or bmyf cDNA to the trp E promoter of E. coli, high level expression of bmyf protein can be obtained in bacterial culture system. Alternatively, in cultured insect cells, bmyf
In order to produce a desired amount of protein, a baculovirus expression system using a known highly active promoter of virus coat protein gene is used. bmyf
Purification of proteins is accomplished by combining techniques known in the art. In the expression system,
The desired protein is the major expression product. The protein is identified by size on an electrophoretic gel. This protein can also be identified and analyzed by antibodies. Such antibodies can be prepared, for example, by immunization of proteins in gel bands. Alternate subregions of 40-80 amino acids in length are synthesized using standard methods known in the art. Alternatively, the specific antibody can also be obtained by inoculating an extract of expressed cells or synthetic peptides into a suitable host (rabbit or mouse), and adsorption of non-bmyf antibody to the control extract of expression cells without bmyf protein. Antibody preparations are removed. The bmyf protein is purified by affinity adsorption using an antibody bound to a solid support material. Protein purification techniques known in the art are optionally utilized to obtain the desired purity. The purified bmyf protein is then characterized by standard methods.

Example 7.1 pMMTV-bmyf and pMMTV-my
Stable Cell Lines Transfected with the oD Construct The pMMTV-bmyf and pMMTV-myoD constructs shown in Example 6.4 were used in RSVne in C3H10T 1/2 and NIH3T3 cell lines (available from American Type Culture Collection).
cotransfect with (Gorman, C (1985) DNA
Cloning: A practical Approach II (Glover, D) 143
-190) and stable cell lines expressing bmyf and myoD under steroid-induced conditions are isolated. Transfection was performed by the calcium phosphate method
(Clark, T. et al Nucleic Acids Res (1990) 18 : 3147-315
3; Glover, DMDNA Cloning; A Practical approachII
(1985) 143-190). These cell lines have been previously confirmed to be dexamethasone-inducible in myoblast formation and bmyf transcript expression as analyzed by Northern blot. These cell lines are used to analyze the turnover rate of bmyf transcripts. In addition, the bmyf protein produced and purified from these cell lines can be further characterized by, for example, bmf.
Used for analysis of yf protein phosphorylation status and localization.

Example 8 Antibody Polyclonal and monoclonal antibodies against bmyf protein can be obtained by using bmyf protein as an antigen. The methods and techniques utilized are those routinely used in the art for immunization, antibody selection, purification, hybridoma screening and immunoassays. Selection of such a combination of methods, as is routinely practiced by one of skill in the art, results in structural and functional properties of the bmyf protein as established in the previous examples, including immunoassay, staining, inactivation, affinity. Antibodies adapted to known techniques such as purification, epitope mapping, etc. are produced and purified. Typical antibody preparations are proteinaceous compositions containing either polyclonal or monoclonal antibodies, whether polyclonal or monoclonal, depending on the method used to obtain the antibodies. Antibodies specific for the bmyf protein are understood in the field of immunology to be antibodies that bind to bmyf. Since the bmyf protein has a homologous portion with other proteins, some antibodies specific to the bmyf protein may cross-react with other proteins to some extent. Antibodies that are tolerant of cross-reactivity for some purposes, but do not undergo measurable cross-reactivity with other proteins for other purposes are preferred.

Applicants are numbered 1-19, 101-118 and 154-1
Rabbit polyclonal antibodies were raised against three synthetic peptides containing the amino acid at position 69 (relative to the N-terminus). First, the region that seems to have antigenicity is PEPTIDESTR.
UCTURE ^TM Program (Jameson, BA & Wolf, H. (198
8) Comput. Appl. Biosci. 4 : 181-186; Pearson, WR &
Lippman, DJ (1988) PNAS USA 85 : 2444-2448; available from University of Wisconsin, Madison) and selected using a region with an antigenic index of at least 1,000. Antigen index can be calculated as hydrophilicity, surface accessibility, type of structure (eg, α,
β or β sheet) and Chou-Fas
man) is a compilation of peptide parameter information including characteristics. Furthermore, the selection of the antigenic peptide used to generate the antibody is (1) the length of the antigen region, (2) the presence of a strong β-fold structure in the predicted surface region, (3) the antigen region. The absence of cysteine in (4)
It was performed based on the absence of known homology with other proteins.

It has been found that an antigenic region of at least 15 residues is preferred. Because they are likely to present the antigenic region within a putative conformation in vivo. Also, the longer fragment potentially provides more epitopes recognized by the antibody,
This makes it easier to obtain specific antibodies. The presence of strong β-folding structures in the expected surface area is preferred. This is because the strong folded structure has often been found to retain its in vivo conformation also in vitro. The absence of cysteine in the antigenic region is preferred as it avoids reaction of the thiol groups with the reagents used to immobilize the antigenic peptide on the particles. The presence of cysteine in the region located adjacent to the antigenic region may be useful in anchoring the fragment to particles or supports. Cysteine can be inserted if it is not naturally present in the flanking region. To avoid such antibody cross-reactivity with other proteins, it is preferred that the antigenic region is heterologous to the other proteins. TFASTA program (Pearson, WR '
Lippman, DJ (1988) PNAS USA 85 : 2444-2448; available from University of Wisconsin, Madison, Wis.) Was used to translate the sequence of the antigenic region from the nucleotide sequence of EMBL registered in the Genebank database. All amino acid sequences obtained were compared. To avoid cross-reactivity with other proteins, the antigenic region used to raise the polyclonal antibody shares homology with all possible amino acid sequences translatable from the gene bank nucleotide sequence. Selected based on not being. When the above analysis was performed, the mouse myo D
Only (not bovine myo D) was obtained in the Genebank.

For each peptide selected, antisera were generated by the following method: (1) intramuscular administration of 50 μg of peptide mixed with complete Freund's adjuvant into rabbit muscle, (2) after 2 weeks, Intramuscularly administering 100 μg of the peptide mixed with incomplete Freund's adjuvant, (3) intramuscularly administering 100 μg of the peptide mixed with incomplete Freund's adjuvant weekly for 3 consecutive weeks, (4) ) Leave for 3 weeks, (5) Boost with 100 μg of peptide mixed with incomplete Freund's adjuvant, (6) Leave for 3 weeks,
And (7) mixed with incomplete Freund's adjuvant
Boost with 100 μg peptide. Serum was then collected and the reaction with bmyf and bovine myoD determined by immunoprecipitation.

It was found that each antiserum reacts with bmyf protein and cross-reacts with myoD protein in each antigenic peptide containing amino acids 1-19, 101-118 and 154-169. The polyclonal antibody precipitate can be further purified to reduce or eliminate antibodies that cross-react with myoD. This purification can be accomplished by previously known methods such as the antibody clearance method by affinity chromatography using myoD immobilized on a substrate. For example, the antibody preparation can be passed through a chromatography column in which the mammalian myoD protein or related proteins, or antigenic fragments thereof (including synthetic N-terminal fragments) are immobilized on a solid support or substrate, Results Antibodies reactive with myoD attach to the column. If desired, antibodies that are cross-reactive with other myogenic proteins can be removed as well. The eluate produced contains a high proportion of antibodies that are specific for the bmyf protein and do not react with bovine myoD.

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[Brief description of drawings]

FIG. 1 shows a recombinant clone λD11 Bmyf.
2 is a restriction map of the bmyf gene in 1 and sequences flanking on both sides.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 8, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Detailed explanation of the invention

[Correction method] Change

[Correction content]

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to cloning, expression, expression products, regulation and genetic engineering of genes involved in differentiation of progenitor cells into muscle cells (myogenesis).

[0002]

BACKGROUND OF THE INVENTION The present application is a priority claim application based on a part continuation application of a co-pending US patent application filed on December 7, 1990 (application number not yet attached). Is. The U.S. patent application filed on December 7, 1990 is a partial continuation application of U.S. Patent Application No. 454,080, which was filed on Dec. 21, 1989, and which belongs to the affiliation.

(Myogenesis at the Cellular Level) Myogenesis can be divided into three general stages (edited by Emerson, C. et al., 1986, Molecular Biology of Muscle Developm).
ent , Alan R. Liss, Inc. NY). First, mesodermal cells are transformed into proliferating myoblasts. Second, myoblasts fuse to form multinucleated myotubes. Muscle-specific genes are activated in this myotube. In the first decision stage, the myogenic lineage is taken over by the cell and all its progeny. In the second terminal differentiation stage,
Myoblasts form non-dividing myotubes that activate a set of muscle-specific genes. In the subsequent third stage, a complex series of regulatory events accompanies maturation in which various muscle protein isoforms are formed. Since proliferative myoblasts themselves can be identified as subclones that take over the activation of the embryonic heavy chain myosin gene that is either fast-twitch or slow-twitch,
Seems to be different.

Although the step leading to the origin of mesodermal cells was not involved in this pathway, many details of the cellular pathway to skeletal muscle have been studied in the early embryo. Skeletal muscle is derived from the middle layer, ie, the mesoderm layer, out of the three germ cell layers (the surface layer, the inner layer, and the mesoderm layer) that are organisms. Mesoderm is a pluripotent tissue that differentiates into many cell types except for skeletal muscle, including: That is, the cell types include bone, cartilage, and myocardium; eyes, teeth, kidneys and parts of the gonads; and a large number of cells in the hematopoietic pathway (red blood cells, lymphocytes, macrophages, etc.). The mesoderm is also identified by where it is formed in various regions of the early embryo (dorsal, head, lateral), and it is the dorsal region that produces morphologically visible somites and sarcomerees. The germ layer. Skeletal muscle is differentiated from these somites and sarcomere. Thus, the decision involves the conversion of dorsal mesoderm cells to proliferative myoblasts, resulting in the inheritance of myogenic lineages by mesoderm cells and their progeny.

An industrial concern for genes that regulate myogenesis is their use in promoting recovery of muscle damage. Muscle contains satellite cells, which are dormant myoblasts. The ability to distribute satellite cells to the site of injury and differentiate into myotubes may lead to the development of new medicines that increase the rate of healing of injuries, especially muscle trauma. Other industrial concerns are focused on livestock. This is because, in this field, the use of a myogenic gene can improve productivity with high efficiency and reduce the use of feed additives. At present, additives such as estrogens and antibiotics are widely used to achieve maximal muscle gain and weight gain. However, these additives may remain in human food and cause the growth of microorganisms resistant to antibiotics,
Its use is often criticized. Myogenic genes can also act on livestock before and after birth to reduce fat accumulation and increase myogenesis. As a result, lean meat (eg, low in fat and cholesterol)
Or healthier human food. Many substances developed from myogenic genes have commercial diagnostic uses in veterinary and human medicine.

Recently, mouse C3H10T1 responds to an effective inhibitor of DNA methylation (5-azacytidine).
Significant advances have been made in the isolation of genes involved in establishing muscle cell lineages by exploiting the unusual properties of cells. After treatment with this agent, colonies containing polynuclear myotubes form in few survivors (Constantides,
PG et al. (1977) Nature 261 : 364; Taylor, SM and J.
ones, PA (1979) Cell 17 : 771; Jones, PA and T
aylor, JM (1980) Cell 20 : 85). By isolating unfused myoblasts, stably growing and inducing
Myotubes in which muscle-specific genes are activated (eg, myosin H chain, myosin L chain (lemb, 1, 2, 3),
Differentiate into α-actin, α, β-tropomycin, and tropomycin C and T) (Konieczny and Eme)
rson (1984) Cell 38 : 791-800).

These observations indicate a simple “master gene” hypothesis (Konieczny, S. in Emerson et al., Supra).
(See F. et al.). This hypothesis is based in part on the fact that genes that are slightly methylated for cytosine residues are normally actively transcribed, whereas genes that are highly methylated are transcriptionally inactive. .. According to this model, a single gene (eg,
Myogenic determination gene) is activated by demethylation reaction,
10T1 / 2 cells are transformed into proliferating myoblasts that can differentiate into myotubes. When DNA derived from myoblasts (quail and mouse) and not derived from non-muscle cells is introduced into 10T1 / 2 cells by transfection,
This model was confirmed because it was shown that these 10T1 / 2 cells could be converted to myoblasts (Konieczny, SF et al. In Emerson et al., Supra; Lassar, A.
B. et al. (1986) Cell 47 : 649-656). Also, the unmethylated human DNA, were able to induce 10T1 / 2 cells myogenic lineage (Pinney, DF et al. (1988) Cell 53: 78
1). If DNA from widely distributed species (ranging from birds to humans) functioned similarly in the transfection assay, gene function was significantly conserved.

(MyoD gene lineage) As an analysis method,
Using transfection of 10T1 / 2 cells with DNA, myoD (Davis, RL et al. (1987) Cell 51 : 98
Two different genes named 7) and myd (Pinney, DF et al., 1988) were first identified. These genes transform 10T1 / 2 cells into myoblasts capable of forming myotubes. cDNA for myoD1 is 10
It has been cloned from a subtracted cDNA library from T1 / 2 myoblasts and sequenced (Davis et al., 1987). Its myogenic activity was analyzed as a chimeric gene using a retroviral promoter. myd has not yet been cloned. According to the hybridization standard in Southern blot, m
yoD and myd are not related to each other (Pinney
Et al., 1988). Biochemistry research shows that myoD is 30-6
It has been shown to be a nuclear phosphoprotein that converts with a half-life of 0 minutes (Tapscott, SJ et al. (1988) Science, 242 :
405). The transcript is also unstable (Thayer, MJ et al. (19
89) Cell 58 : 241).

[0009]

SUMMARY OF THE INVENTION A novel gene involved in the regulation of myogenesis (in the present application, its expression product, bovine myogenic factor (bm
(named for yf)) was isolated as a cDNA and characterized.

The bmyf cDNA was, unexpectedly, isolated in a screen for sequences homologous to myoD. The present invention provides a bmyf coding sequence, bmy
fcDNA, bmyf genomic DNA, promoter of bmyf gene, bmyf protein, polyclonal and monoclonal antibodies against bmyf protein,
Provide a chimeric gene construct of a bmyf promoter and a heterologous gene to be regulated so that bmyf is regulated in vivo, and a bmyf coding region that expresses bmyf under conditions other than those that normally induce bmyf It is a thing. The cDNA encoding bmyf has been cloned into bmyf in various art-recognized expression systems.
It is useful for large-scale synthesis of f protein. This cDNA is also useful for constructs that improve muscle maintenance, or for genetic modifications that increase weight gain and meat production efficiency. For example, a transfected cell line containing the above chimeric gene construct can be used to screen for compounds that can affect myogenesis.
The bmyf genomic gene is useful as well, but is particularly useful for gene replacement and gene therapy applications. bmyf
Promoters are particularly useful as regulators of reporter genes that are desired to be activated or repressed in contact with other myogenic genes. This promoter is also useful for analytical systems that measure the effects of compounds as potential regulators of myogenesis. Screening for compounds that affect myogenesis as therapeutic agents or regulators of muscle development can be performed using the construct in which the bmyf promoter and an appropriate reporter gene are linked. These constructs are DNA
It can be introduced into various cell lines by transformation. Transformed cells contain bmyf protein or b
It is a model for screening compounds that affect the activity of the myf promoter. Although the bmyf protein itself acts as a pharmacological agent, it is more likely to be used as a model for the production of smaller molecules with similar effects. In addition, bmyf protein is
It is useful as an antigen for producing and selecting polyclonal and monoclonal antibodies against bmyf. The antibody itself is useful as a diagnostic tool to analyze bmyf in cells under normal and pathological conditions. The effect of treatment with various drugs on the bmyf promoter or protein can be monitored by an antibody against bmyf. Monoclonal antibodies against various bmyf anti-determinants are useful in mapping the functional domains of the bmyf protein. The chimeric gene construct of the bmyf promoter and the heterologous coding sequence provides a test system for determining the effect of various compounds on normal and pathological bmyf expression as well as the transfected cell lines. Chimeric gene constructs with an exogenous promoter and a coding sequence for bmyf (cDNA or genomic sequence) are useful for therapeutic replacement in the presence of genetic conditions characterized by aberrant expression, or for example in feed It is useful to provide higher expression levels and increase meat production under the control of external stimulants such as additives. The above is illustrative of the applications in which the products of the present invention should be used and is not a complete listing of the applications.

Those skilled in the art can use the well-established and characterized methods known in the art for bmyf cD
Based on the availability and characterization of NA, various products of the invention can be isolated and used to characterize.
For example, bmyf cDNA serves as a probe to isolate genomic bmyf DNA from a bovine genomic library. Many expression systems or baculovirus eukaryotic expression systems that express bmyf cDNA to produce bmyf protein, including fusion proteins to bacterial trpE gene proteins, are known in the art. By isolating the bmyf genomic clone, the elements that control the transcription of the bmyf gene, including the promoter of the bmyf gene, can be identified. After sequencing by well known methods, this promoter can be used to ligate with a heterologous coding sequence to construct a chimera. With the disclosed cDNA coding sequences, one of skill in the art can also construct chimeric genes in which exogenous or heterologous promoters, such as those of retroviral or muscle-specific genes, control the expression of the bmyf coding sequences. If the whole bmyf protein or various subregions provided by peptide synthesis are used as antigens,
Well-known methods for producing polyclonal or monoclonal antibodies are available. Accordingly, one of ordinary skill in the art, given the data, information, and teachings disclosed herein, can readily utilize various products of the present invention. Alternatively, the activity of the bmyf promoter or polypeptide may be increased or decreased, or the bmyf polypeptide or m
Well-known techniques for increasing or decreasing RNA stability can be used to introduce conservative nucleotide sequence changes (changes that do not result in a modified amino acid sequence) into a DNA sequence.

The present invention relates to bmyf protein or bmyf
Provided is a cloned DNA (eg, cDNA, genomic DNA) containing a nucleotide sequence encoding an active protein. The present invention also provides DNA containing the promoter of the bmyf gene. Furthermore, the present invention provides such cloned DNA or cultured cells transformed with such DNA, purified protein having an amino acid sequence containing the amino acid sequence of bmyf active protein, polyclonal antibody or monoclonal antibody specific to bmyf protein,
And a proteinaceous composition comprising these polyclonal or monoclonal antibodies.

[0013]

DETAILED DESCRIPTION OF THE INVENTION Generally, the terms used in this application are standard terms understood by those of ordinary skill in the art of molecular biology, genetics, and cloning. Some terms are further defined for greater clarity. Scientific journals in the above fields (Nature, Cell, Proceedings of the Nationa
Standard abbreviations accepted by the Academy of Science USA, Science, etc.) are used.

The term myogenesis is a term used in the multi-step process of cell differentiation in which non-muscle stem cells are converted into muscle cells. Myogenesis is the production of myoblasts, mRNA or protein of muscle-specific genes (H and L chains of myosin, α-actin, α-tropomyosin and β-tropomyosin, and troponin C and troponin T).
Expression, myotube formation, fusion cell generation, and muscle-specific promoter activity are recognized by any of several criteria. The currently available analytical method for the early stages of myogenesis uses the publicly available embryonic mouse fibroblast cell line C3H10T1 / 2. C3H10T1 / 2 cells undergo myogenic conversion by transfection with an expressible myogenic gene such as MyoD or by treatment with 5-azacytisine. In the present invention, C3H1 by bmyf cDNA in an expression vector such as pEMSV-bmyf.
It is shown that transient transfection of 0T1 / 2 cells elicits a myogenic response in C3H10T1 / 2 cells.

The term promoter is used to indicate a gene region that has the function of controlling the transcription of a gene. The promoter is typically 5'of the coding region
It is located near the end (the region encoding the amino-terminal portion of the encoded protein). As is known in the art, the location of the promoter is based on the genomic DNA
Sequence analysis and other approved methods (S1 mapping method, DNAseI footprinting method, gel delay analysis method, and transient transfection method with promoter and reporter gene). Promoters include sequences that vary in size and have more than one regulatory function (eg, regulatory functions with responses to inducers, repressors, and activators and tissue specificity, etc.).

The bmyf active promoter is a variant of the nucleotide sequence of the bmyf promoter sequence,
It has at least 80% homology to the yf promoter sequence and has substantially the same function as the bmyf promoter. The bmyf promoter controls the transcription of the bmyf gene, has an RNA polymerase binding site,
It contains a regulatory region that activates and / or represses the transcription of the yf gene. The regulatory region may be located 5 ′ to the structural gene, within the structural gene (including introns), and / or 3 ′ to the structural gene. As discussed in the Examples below, the regulatory region of a promoter and its function can be identified by methods well known in the art.

The term coding region is used to indicate the part of the gene that codes for a protein. cDNA
In, a single polypeptide chain is encoded by a continuous uninterrupted coding region. In genomic DNA (eg, the gene itself), its coding region is usually interrupted by one or more introns. These introns are non-coding intervening sequences present in the gene, but not in mRNA.
A coding region is under the control of this promoter if it controls the expression of the protein encoded by the coding region.

Genomic DNA is DNA that is directly cloned from the chromosomal DNA of an organism. The bmyf gene is a genomic D cloned from the chromosomal DNA of an organism.
Included in NA. Thus, genomic DNA is
Before transcription, shows the structure of a gene in the chromosome of an organism.

The term expression means that the presence of a gene in a cell or organism becomes detectable. Expression occurs whenever a phenotype attributable to the gene is observed. More directly stated, gene expression is the synthesis of a gene product by transcription into mRNA or translation of mRNA into protein.

The term chimeric gene is used to indicate an artificial gene constructed by joining together functional genetic elements obtained from different sources. For example, bmyf
A chimeric gene can be constructed by linking a promoter to the coding region of another gene. Such coding regions can be found in bmy in naturally occurring cells.
It is called the heterologous coding region because it is not regulated by the f promoter. If the heterologous coding region is expressed to provide a readily detectable product, the chimeric gene is used as a reporter gene to measure the activity of the promoter under experimental conditions. Another example of a chimeric gene is a gene constructed by joining an exogenous or heterologous promoter with the bmyf coding region. The exogenous promoter is
It is a promoter that does not regulate the bmyf gene in naturally occurring cells.

The bmyf protein is an expression product of the bmyf gene. The deduced amino acid sequence of the bmyf protein is shown in SEQ ID NO: 3.

The bmyf active protein is an amino acid sequence variant of the bmyf protein having at least about 80% sequence identity with the bmyf protein and having substantially the same activity. Therefore, the bmyf protein is
It is known to function as a regulatory protein that plays a fundamental role in controlling myogenesis in mammalian cells. Further studies will confirm its function more quantitatively and clarify the molecular mechanism of its activity. It is understood that many amino acid sequence variants are possible that have substantially the same activity. Such variants include, but are not limited to, conservative amino acid substitutions as well as substitutions and deletions in non-critical regions of the protein. Such variants are called bmyf active proteins, as long as they have substantially the same activity as bmyf protein itself (distinguishable from other proteins of myogenesis and quantitatively similar), Included within the scope of the invention.

The methods used herein are either specifically cited or are well known and are available in several publications which summarize the methods. Publications include, for example, Maniatis et al., Molecular Cloning, a Labor.
atory Manual , Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York (1982); Genetic Engineerin
g , Plenum Press, Ner York (1979); Weir (1989),
Of the four volumes, Handbook of Experimental Immunology , Fourth Edition, Blackwell Scientific Publications, Oxford;
And many volumes in Methods in Enzymology , Acad
There are emic Press Press and New York.

Various cell lines and plasmid vectors have been disclosed. All are publicly available. bmyf
Cells or tissues that serve as a source of cDNA or bmyf genes are readily available. The plasmids, cloning vehicles, and cells used for clonal transfer and expression were chosen for ease of manipulation. Substitutes can be used at any stage, paying attention to the desired properties, according to criteria and properties known to those skilled in the relevant art.

Examples of the present invention will be described below. In these examples, the bmyf cDNA of the invention is
And the cloning and characterization of other products.

Example 1 Construction of cDNA Library and
Gosp except that selection by screening cytochalasin was omitted
Bovine myogenic cells were isolated from fetal thigh muscle as described by odarowicz et al. (1976) J. Cell. Biol. 70 : 395-405. Cells were treated with 4M guanidine thiocyanate, 0.5% (v
/ v) Sarkosyl, 0.025M EDTA, and 0.1M β-mercaptoethanol were used for dissolution. Chirgwin et al. (1979) Bioch
emistry 18 : 5294-5299; Freeman et al. (1983) Proc.Natl.Ac
Cs as described in ad.Sci.USA 80: 4094-4098.
RNA was isolated by precipitation of Cl. RNA pellet for 10
mM Tris ・ HCl (pH7.6), 1 mM EDTA, 5% (v / v) Sarkosyl,
And dissolved in 5% (v / v) phenol and then extracted with phenol: chloroform: isoamyl alcohol (50: 49: 1). After ethanol precipitation, RNA was dissolved in deionized water and stored at -70 ° C. Poly A ⁺ RNA was isolated by chromatography using oligo (dT) -cellulose,
Used to synthesize first-strand cDNA with AMV reverse transcriptase and oligo (dT) as primer (Berger and Kimmerl (1987) Meth. Enzymol. 152). Second-strand synthesis was performed using DNA polymerase I under storage of RNase H (Gubler (1987) Meth. Enzymol. 152: 330-335). CL
Chromatography with -4B Sepharose
CDNAs with an average length of 500bp or more were isolated, methylated, and Ec
It was ligated to the oRI linker at the blunt end. The cDNA bound to the linker was cleaved with EcoRI, fractionated according to size, and
It was cloned into the EcoRI site of gt10. Commercially available extract (Pack
^{ageneR TM system; Promega, Medison,} WI) using, after the recombinant phage DNA was packaged, to amplify the library.

A phage library containing about 3 × 10 ⁴ recombinants was used as a template for pVZC11b plasmid DN.
Using a ³² P-labeled 274 bp fragment of mouse MyoD1 cDNA amplified by polymerase chain reaction (PCR) using A,
Thorough screening (Sakai et al. (1985) Science 23
0: 1350-1354; Mullis and Faloona (1987) Meth, Enzymol,
155: 335-350). A pair of degenerate 1's starting at nt339 and 612 of the sense and antisense cDNA strands, respectively.
An 8-mer primer was synthesized and used to amplify this 274 bp fragment. This fragment spans the cys / his region, the basic region, and the myc homology region of MyoD1 and is randomized to oligonucleotide priming (Feinberg and Vogelstein (1983) A
nal.Biochem. 132: 6-13), high specific activity (10 cpm / μg or more) was labeled with ³² P- (α) dATP. For screening libraries, standard methods (Car
plaques were bound to nylon filters (Micron Separations, Inc) using lock (1986) Focus 8: 6-8).
Heat these filters at 70 ° C for 60-90 minutes and
× SSC (0.9M NaCl, 0.09M sodium citrate, pH 7.0),
0.05% (w / v) SDS, 0.25% (w / v) Blotto, 1 x Denhardt's solution (0.02% (w / v) Ficoll, 0.02% (w / v) Polyvinylpyrrolidone, 0.02% (w / v) BSA), 0.05% (w / v) sodium pyrophosphate, and 5 mg / ml denatured salmon sperm DN
A was prehybridized at 50 ° C. for 6 to 12 hours.
Hybridization is 6 × SSC, 0.5% (w / v) SDS,
And in a buffer containing 0.05% (w / v) sodium pyrophosphate at 50 ° C. for 24 hours with 10 cpm / ml denaturing probe. Next, the filter is 2 × SSC, 0.1%
(w / v) 2 times at room temperature in SDS, 1 × SSC, 0.1% (w / v) SD
Once in S at 54 ℃, and 0.5 × SSC, 0.1% (w / v) SD
Washed once in S at 54 ° C. Each wash lasted 15 to 30 minutes. After exposure of the filters to Kodak X-0MAT film, positive recombinants were purified by serial dilution and rescreened under the conditions described above (Carlock et al., 1986).

Example 2 Characterization of cDNA clones Recombinant phage DNA was cleaved with BamHI and electrophoresed on a 0.7% agarose gel, 1M ammonium acetate and 0.02N NaOH (Rigaud et al. (1987) Nucl. Acids Res. 15 : 8
57) in Biotrans nylon filter (ICN Biomedicals
Inc, Costa Mesa, CA). The filters were UV-irradiated, heated at 70 ° C. and hybridized to the 274 bp probe, essentially as in the library screen above. Recombinant insert, pGEMTM-3zf (-)
(Promega) was subcloned into the EcoRI site to create a restriction endonuclease map. Various subfragments are M13
Subcloned in and sequenced on both strands by the dideoxy method of Sanger et al. (1987). The restricted part is
Especially when it is missing in the 3'untranslated region of bmyf,
A 17mer was synthesized to complete the sequencing.

The complete nucleotide sequence of the bmyf insert is shown in SEQ ID NO: 2. This nucleotide sequence is
It contains 1,889 bases (excluding the short poly A portion) and contains a long open reading claim of 765 nucleotides beginning at the potential ATG start site at nucleotide 69. The deduced amino acid is a protein derived from the longest open reading frame of human myf-5 cDNA (Braun et al. (198
9) It can encode a protein of 255 amino acids with the same size as EMBO J.8: 701-709). bmyf and human myf have extensive homologous regions, especially in their coding regions. 96% identical at the amino acid level and 92% identical at the nucleotide sequence level. Both cDNAs contain a short segment (68 in bmyf) upstream of each potential ATG start codon.
Nucleotides, 42 nucleotides in human myf-5).
These regions are also highly related, exhibiting 82% sequence conservation. Although it is unclear whether the first nucleotide is the actual 5'transcriptional start site of bmyf mRNA, a separately cloned smaller bovine cDNA (clone 7) contains the same nucleotide containing the first nucleotide. It has a 5'side sequence. This suggests that bmyf is indeed a full-length cDNA.

The main difference between bmyf and human myf-5 is that bmyf
Has a 3 ′ untranslated region that is longer by several hundred nucleotides. From the stop codon, AA at nucleotide 1407 in human myf-5 and at nucleotide 1415 in bmyf
2 up to the TAAA polyadenylation signal
The two cDNAs are very related and 81% of the sequences are similar. Human myf-5 terminates within a short distance of this polyadenylation signal, while bmyf extends an additional 475 nucleotides, the second at nucleotide 1872.
It terminates near the polyadenylation signal of. Probably
Human myf-5 cDNA is derived from a transcript that terminates at upstream sites, whereas bmyf is believed to be derived from a transcript that terminates at downstream sites. As described below, RNA from fetal skeletal muscle contains a 1.5 kb bmyf transcript that is believed to result from adenylation at the AATAAA site at nucleotide 1415.

Example 3 Northern Hybridization
It was excised various tissues from emission analysis 20cm fetal bovine and frozen in liquid nitrogen. Skeletal muscle was taken from the hind and forelimbs. To prepare RNA, frozen tissue was ground into a fine powder and 5M guanine thiocyanate, 10 mM EDTA, 50 mM Tris-HCl (pH 7.
5) and 8% (v / v) β-mercaptoethanol. RNA was precipitated directly from the lysate using 4M LtCl as described above (Cathala et al. (1983) DNA 2: 329-33.
Five). After solubilization and extraction with phenol, RNA was precipitated with ethanol to give LiCl pellets or an aqueous solution.
Stored at 70 ° C (Cathala et al., 1983). Poly A ⁺ RNA,
Isolated as described above, electrophoresed on 1.1% agarose formaldehyde and transferred to Biotrans nylon membrane in 20xSSC. The filter was irradiated with UV, heated at 70 ° C. for 60 to 90 minutes, and hybridized with ³² P-labeled cDNA as follows. 0.5M sodium phosphate (pH7.2), 7% SDS, 1
Prehybridize the filters in% BSA and 1 mM EDTA at 65 ° C for 4 hours, then use base-denatured probe (10 ⁶ cpm / ml or more) under the same conditions for 18-2.
Hybridized for 4 hours (Mahmoudi and Lin (198
9) Biotechniques 7 : 331-333). Use a 40 mM sodium phosphate buffer (pH 7.2), 5% (w / v) BSA, 5% (w
/ v) SDS and 1 mM EDTA at 65 ℃ for 30 minutes each, 2
Washed twice, 40 mM sodium phosphate (pH 7.2), 1 mM EDTA,
And washed twice in 1% (w / v) SDS at 65 ° C (M
ahmoudi and Lin, 1989). The probe was gel purified before use (Dretzen et al. (1981) Anal. Biochgem 112 : 29.
5-298).

When skeletal muscle-derived poly A ⁺ RNA was analyzed by Northern blot using the full-length bmyf cDNA as a probe, it was detected that three kinds of RNA migrated to 1.5 kb, 2 kb, and 3 kb. The 1.5 kb band is most abundant and is present at levels 2-3 times higher than the high molecular weight species. The presence of three different bands in this Northern blot indicates that bovine
It shows that transcripts from the myf gene are processed separately. For example, the 1.5 kb RNA band is equivalent to the transcript that produces human myf-5 cDNA, and may be lacking the region downstream of the proximal polyadenylation signal of bmyf at nucleotide 1415. To investigate this, Northern blots of poly A ⁺ RNA from bovine skeletal muscle were derived from the distal end of bmyf at nucleotide 14
354 bp, 3'to the polyadenylation signal at 15
It was probed with the AvaII / EcoRI fragment. The distal 354bp fragment is 2
Hybridized with kb and 3 kb bands, but 1.5 kb
Substantially did not hybridize with RNA. This indicates that this species lacks the same portion of the missing 3'sequence of the human myf-5 cDNA.

To examine whether bmyf is expressed in a tissue-specific manner, poly A ⁺ RNA derived from several fetal bovine tissues was examined.
Was subjected to Northern blot analysis and probed with the full-length bmyf cDNA. Only RNA from skeletal muscle gave a signal. By comparison with the signal produced by known amounts of bmyf RNA synthesized from the pG EMTM vector containing the bmyf insert, the actual level of bmyf transcript present in skeletal muscle was low, 0.001% to 0.0% of total message.
It is estimated to be 001%. RNA from the mouse azamyogenic cell line
When subjected to Northern blots at the level of stringency used above, little or no code was detected.

Example 4 Cell culture and DNA transfer
CHT obtained from Ekushon American Type Culture Collection
1/2 (clone 8), 10% heat inactive FCS, 100 μg / m
l DMEM (Cellogo ^TM , Mdiatech, W
(A. Washington, DC). A stable azamyogenic cell line was obtained by treating C3H10T1 / 2 cells with 3 mM 5-azacytidine for 24 hours and serially subcloning them (Konieczny and Emerson, 1984). When plated at low density, over 90% of the colonies derived from the 91a and 56a azamyogenic subclones were positively stained for a monoclonal antibody against the heavy chain of skeletal myosin (MF-20, see below). A large number of multinucleated fused cells were formed.

DNA transfection was carried out using pEMSV-scrib
This was done using the Moloney sarcoma virus LTR of e and a cDNA inserted at the EcoRI site between the SV40α polyadenylation signal (Harland and Weintraub (1985) J. Cell.
Biol. 101 : 1094-1099; Davis et al., 1987). Full length bmy
The f cDNA was inserted in both sense and antisense orientations. In transient transfection (Wright et al. (1989) Cell 56 : 607-617), C3H10T1 / 2 cells were
1 × 10 ⁵ cells were plated per 10 cm dish and grown in DMEM containing 10% heat-inactivated fetal bovine serum for 2 days. Replaced 3 hours before transfection and described (Gorman
(1985) DNA Cloning VolII, IRL Press, Oxford UK, p
p.143-190), calcium phosphate precipitates (7.5
Plasmid DNA was added to μg / dish). The cells were incubated for 24 hours and then phosphate buffered saline (137 mM NaCl,
Wash with 2.7mM KCl, 4.3mM Na HPO, and 1.4mM KH PO) and differentiate with differentiation medium (D containing 2% (V / V) heat-inactivated horse serum)
They were maintained in MEM) or grown in normal medium for 2 days before incubation in differentiation medium. Three
After a day, the cells were treated with 70% (V / V) ethanol, 3.7% (V / V)
Immobilized in formaldehyde and 5% (V / V) glacial acetic acid, and then reacted with a monoclonal antibody against the heavy chain of skeletal muscle myosin (MF-20, Bader et al., 1982) and then bound to alkaline phosphatase. Goat anti mouse Ig
G was made to react. For immunostaining, NBT and BCIP staining reagents
(Promega) and following the supplier's instructions.
Micrographs were taken with a Kodak Panatomic-X film using the Z
Photographed with an eiss inverted microscope.

A transient assay using the same vector
Bovine myf cDNA was transferred to C3H10T by transfection.
It shows that 1/2 cells can be converted to myoblasts. b
With the above expression vector having myf in the sense direction, C3H10T1 / 2
Cells were transfected, induced to differentiate for 3 days, then fixed and even reacted with a monoclonal antibody against the heavy chain of skeletal muscle myosin. Cells were transfected with the same vector with bmyf in the antisense orientation or mouse Myo D1 in the sense orientation (as negative or positive controls, respectively). Many cells were positive for antibodies in cultures transfected with the bovine myf expression plasmid in the antisense direction that reacted positively with the myosin antibody probe by transfection with the bmyf expression plasmid in the sense direction. There wasn't. Cells transformed to the muscle myosin phenotype with the bovine myf expression vector were present in both experiments at frequencies of 10 ^-3 and 10 ^-4 . This frequency is several times greater than the spontaneous activation level in 10T1 / 2 cells (Lassar et al.,
1986). It was observed that both mononuclear cells and polynuclear cells stained positive for heavy chain myosin.

Example 5 bmyf gene and its proximal progeny
By comparing the restriction enzyme map of the motor structure bmyf cDNA with the fetal bovine skeleton genomic DNA, it was presumed that an intron was present in the bmyf gene (Fig. 1). To determine the exact location of the intron in the bmyf gene, the bmyf genomic fragment was amplified by polymerase chain reaction (PCR) using a set of sense and antisense bmyf cDAN primers (US Pat. No. 4,683,202). No.). The amplified genomic fragment containing the larger intron (Intron A in Figure 1) was subcloned and the exact location of the intron was determined by partial sequencing using the dideoxy chain termination method. This fragment was predicted to contain an approximately 1 kb intron between cDNA residues 569 and 570. The restriction enzyme map of genomic DNA (Fig. 1) shows EcoR in intron A.
The I and Xba I sites are indicated. The second amplified fragment contained a smaller intron of approximately 300 nucleotides (Fig. 1
Of the intron B).

The overall structure of the bmyf gene is mouse myf-5.
Is very similar to the herculin gene adjacent to (J.
H. Miner and B. Wold, PNAS USA 87 : 1089-1093
(1990)). This similarity is consistent with the idea that the bmyf and herculin genes arise from sequence divergence from older gene duplications in ancestral genes.

To isolate the proximal bmyf promoter,
"Inverse" PCR was used to increase the 5'region of the cDNA. First, fetal bovine skeletal muscle genomic DNA was digested with baI and EcoRI. As mentioned above, the restriction sites for XbaI and EcoRI were determined to be in intron A. next,
By re-annealing this cleavage product under conditions suitable for ligation of circular molecules, a 930 bp circular fragment consisting of an XbaI fragment extending from −218 to +145 of intron A for cDNA was obtained.
A DNA molecule was formed. With two primers starting at +466 for sense and +218 for antisense, the region between the two primers containing the XbaI site was increased by PCR. This region contained the 5'region of the cDNA.

The 281 bp XbaI / PstI fragment derived from the 930 bp fragment was subcloned and sequenced by the dideoxy chain termination method. The sequence revealed that this fragment contained nucleotides 1-67 of the bmyf cDNA, which
A portion of the 930 bp fragment up to the −218 XbaI site is actually a cDNA
It was found to be present on the 5'side of the mouse and represents the proximal promoter.

Example 5.1 Bmyf fragment derived from bovine genomic DNA
Isolation of Genes To isolate fragments containing genomic bmyf DNA inserts, a commercially available bovine genomic library was screened as follows. (1) a fragment containing the bmyf gene is amplified by PCR using the intron A primer, and (2) above.
The amplified fragment of (1) was screened using the full-length bmyf cDNA as a probe.

The 20,000 recombinants of the Stratagene 1 DASH ^™ II genomic library contain a partially degraded fragment of Sau 3A of bovine genomic DNA. This was seeded on each of 10 large plates. Plate culture lysates were assayed by amplification by PCR with the Intron A primer in the presence of the bmyf gene. Four plates were positive and one was thoroughly screened with the full length bmyf cDNA probe.

ΛDII bmyf1 which is one of the positive plaques
Contains an insert of approximately 13 kb, and its partial restriction map (Fig. 1) shows that it contains the bmyf gene. The bmyf gene (about 3 kb) is flanked at its 5'end by a fragment of about 4 kb, and also at its 3'end by about 7 kb.
flanked by kb fragments. This 13 kb clone was confirmed to contain the bmyf gene based on the fact that the 217 nucleotides of the first exon show complete sequence homology with the sequence of the bmyf cDNA.

In order to determine the sequence of the bmyf coding sequence and the promoter portion, various portions of 13 kb were digested with pGEM.
Subcloned into the EcoRI polylinker site of ^TM- 3zf and -zt vectors (Promega). Isolate single-stranded DNA and accelerate the synthetic reaction using the dideoxy chain termination method 20
Sequencing was performed using mer oligonucleotides. The complex of the cDNA with the available genomic sequence is shown as the 5'to 3'sequence in SEQ ID NOS: 4-12 of the Sequence Listing. cD
The NA nucleotide sequence and the corresponding bmyf amino acid sequence are shown in SEQ ID NO: 1. The cDNA nucleotide sequence is shown in SEQ ID NO: 2. The bmyf amino acid sequence is shown in SEQ ID NO: 3. In SEQ ID NOS: 1 and 2, the coding region of the cDNA sequence is shown by triplet codons, and the non-coding region of the cDNA is shown as a nucleotide sequence in blocks of 10.

SEQ ID NO: 4 begins near (but not including) the BamHI site approximately -4500 nucleotides upstream from position +1 of the bmyf cDNA. The length of the sequence of SEQ ID NO: 4 is
It is 385 nucleotides.

SEQ ID NO: 5 begins at the Sac I site (GAGCTC) approximately -2300 nucleotides upstream from position +1 of the bmyf cDNA. The length of the sequence SEQ ID NO: 5 is 815 nucleotides. Between SEQ ID NO: 5 and SEQ ID NO: 6 is an approximately 440 bp unsequenced segment.

SEQ ID NO: 6 is the proximal promoter sequence starting at -946 nucleotides upstream from position +1 of bmyf cDNA. This sequence attaches directly to position +1 of the bmyf cDNA. Based on the sequence information of the promoter region, this promoter is rich in GC and has three TATA motifs (cDNA +1
Present at −158, −114, and −60 nucleotides upstream of position). This motif is the RNA polymerase
TF, a binding transcription factor required for II-mediated transcription
Can serve as a site for IID. 5'determining the TATA box that is thought to be used as the transcription start site
Footprinting of the area will be described later.

SEQ ID NO: 7 is bmyf cDN starting from position +1
It is the part of A and continues up to +569 of the cDNA, from there the genome
DNA intron A begins. The coding region of the cDNA sequence is indicated by the triplet codon. The non-coding region of the cDNA is shown as a nucleotide sequence in blocks of 10.

SEQ ID NO: 8 is a sequence encoding a part of intron A. Intron A is approximately 1000 bp and is not a complete sequence. SEQ ID NO: 8 is intron A
Is 361 bp at the 5'end. The region following SEQ ID NO: 8 is approximately 4
This is an unsequenced region of 00 bp.

SEQ ID NO: 9 is 400b which has not been sequenced
It is 258 bp of intron A downstream of the p region.

SEQ ID NO: 10 is a sequence directly following SEQ ID NO: 9 and starts from the coding region corresponding to the +570 position of bmyf cDNA.

SEQ ID NO: 11 is the entire sequence of intron B. Intron B is +644 and +65 of bmyf cDNA
Located in the genomic DNA in the part between 5. Intron B is
It is 393 bp long.

SEQ ID NO: 12 is the sequence of exon 3. Exon 3 begins at position +646 in the cDNA. This is 1
It is 245 bp long and extends through the poly A attachment site.

Example 5.2. Transcription start site of bmyf gene
Mapping As mentioned above, the proximal promoter region contains three TATA elements from which transcription can be initiated by RNA polymerase II. RNAase protection experiments were performed to identify the most likely starting site.

An antisense RNA probe labeled with ³² P (extending from PstI at +64 of cDNA to XbaI at −218 upstream) was annealed to total RNA from fetal bovine skeletal muscle (18 cm fetus). It was The annealed mixture was cut with RNAse A and TI. The protected double-stranded fragment is then denatured and then separated on a sequencing gel,
Its size was measured by comparison with a series of dideoxy sequencing reactions over the same region. According to this method, the transcription initiation site is in the vicinity of about -185 nucleotides upstream of PstI at the + 64th position of cDNA, or cDN
It was determined to be in the TATA motif in the vicinity of -114 nucleotides upstream from the +1 position of A.

Judging by the intensity of the probe signal, the early fetus is more likely than the later fetus (eg, 18-
The 42 cm fetus) promoter is considered to be highly useful.

Example 6 Chimeric Constructs Subclones of the bmyf gene containing only the Bmyf coding region or other desired regions of the bmyf gene can be constructed. For example, the sequence information provided herein identifies suitable restriction enzyme sites that can be used to construct and clone the desired segment. Other isolation techniques that do not require sequence information for the desired fragment may also be utilized. It especially includes PCR, which is described in US Pat.
No. 4,683,202. Synthetic primers can be constructed that have a sequence that groups together the desired segments, which allows the polymerase chain reaction, such as a portion of the bmyf promoter, bm
Any desired specific segment of the yf gene can be amplified. In addition, the primer sequences can be constructed to induce restriction sites with the desired specificity flanking the amplified segment, as is well known to those of skill in the art.

A chimeric gene construct of a segment of the bmfy promoter linked to a heterologous gene segment, or
It is possible to create a construct for an exogenous promoter that binds to the bmyf coding region, as will be shown later.

In addition, other parts of the bmyf gene can likewise be subcloned and linked into the chimeric functional gene. In this way, specific functional domains of the bmyf gene can be linked to other genetic constructs, or. It can be deleted from the bmyf gene to give rise to a different function (add or remove function associated with the subcloned domain).

Example 6.1. Chimeric bmyf promoter / CA
The functional chimeric gene in which the T gene construct bmyf promoter and the heterologous gene are linked also regulates the transcription of the heterologous gene, just as bmyf itself is regulated. The model code gene is
For example, in the case of a reporter gene that encodes chloramphenicol acetyltransferase (CAT), the resulting constructs are readily available for stimulants and chemical compounds that affect bmyf expression by either stimulation or repression. It will provide an index that can be measured.

To create a rough map of the functional region of the bmyf promoter, two overlapping duplicates of different length
The bmyf promoter part was linked to the chloramphenicol acetyltransferase (CAT) construct gene. These constructs were then transfected into cell lines and cell CAT activity was measured using ¹⁴ C-chloramphenicol.

One construct contained the full length of the 5'flanking portion of the 13 kb fragment, extending from the unique BamHI site of -4.2 kb to the PstI site of +64. 4.2kb promoter / CAT to construct 4.2kb promoter / CAT gene
To construct the gene, the 4.2 kb fragment has blunt ends at the 5'BamHI site and the 3'PstI site. pCAT ^T
M has a blunt end at the HindIII site in the polylinker,
It is then digested with PstI to form a site suitable for directional cloning of the 4.2 kb promoter fragment.

The second construct contains a proximal promoter segment extending from the XbaI site at -218 to the PstI site at +64. 282 bp for the proximal promoter / CAT construct
The XbaI / PstI fragment of E. coli was created by ligating to the pCAT polylinker site between the XbaI and PstI sites.

Each of these constructs can be used to transiently transfect 10T1 / 2 cells with an expression vector such as pEMSV, as shown in Example 4 above. This transient transfection is
It is performed to determine whether the fragment extending from the 4.2 kb BamHI site to the -218XbaI site contains the functional region of the promoter.

Example 6.2. Bmyf promoter cis segme
Useful chimeric bmyf the identification of cement promoter / CAT genetic
Examples To identify the regulatory portion of the progeny bmyf promoter
A construct confirmed to contain a functional promoter region in 6.1. And a series of deletion mutants of this construct were transfected into a cell line and expressed. The series of deletion mutations consist of progressive deletions from the 5'end of the promoter towards the transcription start site.

First, a large number of cell lines were transiently transfected with the full-length promoter / CAT construct in order to find cell lines capable of expressing the CAT gene lacking the bmyf promoter. For example, the myf-5 gene is L6
And C2C12 cells (Wright, W. et al. (1989) Cell, 56 : 607-671; Braun, T. et al. (EMBO J
8: 3617-3615); Peterson, C. et al. (1990) Cell 62: 493-.
502), these are the optimal target cell lines for transfection of the bmyf promoter / CAT construct. further,
Even though certain cell lines are believed not to express the CAT gene that lacks the bmyf promoter, testing for deletions of such cell lines indicates that these regions function as silencers for the bmyf promoter, that is, they are excluded. It becomes clear that the gene is activated only by being ridden.

Next, the full-length bmyf promoter / CAT construct and its deleted derivative were transiently co-transfected with RSV lac Z into selected cell lines (Sambrook, J.
Et al. (1989) Molecular Cloning; A Laboratory Manual
(2nd edition); Webster, J. et al. (1989) EMBO J 8: 1441-4
6) The CAT activity was measured using ¹⁴ C-chloramphenicol. Β-galactosidase activity is measured as an internal control of transfection effects between cell lines (Webster, J. et al. (1989) EMBO J 8: 1441-46). Transfection is performed by the calcium phosphate method.
(Clark, T. et al. (1990) Nucleic Acids Res 18: 3147−
3153; Glover, DM (1985) DNA Cloning: A Practical
Approach II, 143-190).

When the localization of this important part is carried out by analysis for deletions, the cis element can also be identified. The promoter region thought to be required for promoter activity can be confirmed by gel shift in vitro assay using extracts from cell lines known to express the bmyf promoter / CAT construct.

The promoter fragment determined to contain the cis element is subjected to DNAse footprinting in order to accurately elucidate the protected portion. DNAse footprinting is done by preparing two batches of the promoter fragment in question. One of the two batches is a trans-acting regulatory element (Trans
-batch containing cell extract with -acting regulatory element) and vat without cell extract, each fragment having radiolabeled 3'or 5'ends. So that on average there will be one cut per fragment for this batch,
The DNAse I is allowed to react rapidly, and then each batch is subjected to gel digestion to identify areas not cleaved by DNAse I (areas protected by the prepared protein).

Example 6.3 Identification of enhancer region
Use chimeric bmyf promoter / CAT structure Tsukibutsu constructs of Examples 6.1 and 6.3, as can be identified even a fragment believed to contain bmyf enhancer sequences, it can also be further modified. These sequences, like introns, can be located in the 5'and 3'flanks. Enhancers typically exhibit cell type specificity. The pCAT vector can insert such an enhancer sequence. Several unique restriction sites are included downstream of the CAT gene.
These restriction sites include EcoRI and BamHI.
The resulting construct can be transiently transfected into 10T1 / 2 cells and other cell lines by methods described above or known to those of skill in the art, and bmyf
Protein production was measured to determine the cell type specificity of the enhancer fragment in question.

Example 6.4 Chimeric bmyf promoter / β
-Galactosidase gene construct Another heterologous reporter gene used is the β-galactosidase gene. The bmyf promoter / β-gal gene construct was constructed using pCH110 (Hall, C et al. (1983) J. Mol. App.
l. Genetics 2 : 101-109; available from Pharmacia LKB), and pSP72 (Prom cleaved with BamHI / HindIII) by excising the lacZ gene on a BamHI / HindII fragment.
It can be created by inserting the lacZ gene into ega) with orientation. 4.2kb BamHI / Pst I bm
The yf promoter fragment was then transformed into XhoI and Pvu in pSP72.
It can be inserted into the II site by ligation with blunt ends. pSP7
2 contains a bmyf fragment containing the muscle-specific enhancer (4.2 kb
There are 7 restriction sites that can insert (other than the 5'fragment), which are downstream of the site where the lacZ fragment is inserted. These 7 restriction sites are SmaI, KpnI, SacI, EcoR
I, ClaI, EcoRV, and BglII. The β-galactosidase gene is useful as a reporter gene because it exhibits a staining reaction associated with β-gal activity and because antibodies against β-gal are commercially available.

Example 6.5 Chimeric pMMTV-bmyf gene and
And the pMMTV-myoD gene construct , a chimeric gene having an exogenous promoter linked to the bmyf coding region, expresses bmyf under conditions in which the action of the promoter is normally shown. This type of chimeric gene has a sufficient amount of bm for isolation and further characterization.
The yf protein can be expressed.

For example, the EcoRI restriction fragment containing the complete bmyf cDNA and myoD coding region is a pΩ5 vector.
(Morganstern, J. & Land, H. (1990) Nucleic Acids Re
s. 18 : 1068). This vector has an enhancer-free mouse mammary tumor virus (MMTV) promoter that is 20-50 fold inducible by dexamethasone in mammalian cell culture. The bmyf and myoD coding regions are in the sense direction (pΩbmyf + and pΩmyoD +)
And in the antisense orientation (pΩmyfl ⁻ and pΩmyoD ⁻ ).

Example 7 Expression and purification of bmyf in a cell line
The bmyf protein produced can be obtained by combining the chimeric gene of the present invention with any one of known expression systems. For example, by linking the coding portion of the bmyf gene or bmyf cDNA to the trp E promoter of E. coli, high level expression of bmyf protein can be obtained in bacterial culture system. Alternatively, in cultured insect cells, bmyf
In order to produce a desired amount of protein, a baculovirus expression system using a known highly active promoter of virus coat protein gene is used. bmyf
Purification of proteins is accomplished by combining techniques known in the art. In the expression system,
The desired protein is the major expression product. The protein is identified by size on an electrophoretic gel. This protein can also be identified and analyzed by antibodies. Such antibodies can be prepared, for example, by immunization of proteins in gel bands. Alternate subregions of 40-80 amino acids in length are synthesized using standard methods known in the art. Alternatively, the specific antibody can also be obtained by inoculating an extract of expressed cells or synthetic peptides into a suitable host (rabbit or mouse), and adsorption of non-bmyf antibody to the control extract of expression cells without bmyf protein. Antibody preparations are removed. The bmyf protein is purified by affinity adsorption using an antibody bound to a solid support material. Protein purification techniques known in the art are optionally utilized to obtain the desired purity. The purified bmyf protein is then characterized by standard methods.

Example 7.1 pMMTV-bmyf and pMMTV-my
Stable Cell Lines Transfected with the oD Construct The pMMTV-bmyf and pMMTV-myoD constructs shown in Example 6.4 were used in RSVne in C3H10T 1/2 and NIH3T3 cell lines (available from American Type Culture Collection).
cotransfect with (Gorman, C (1985) DNA
Cloning: A practical Approach II (Glover, D) 143
-190) and stable cell lines expressing bmyf and myoD under steroid-induced conditions are isolated. Transfection was performed by the calcium phosphate method
(Clark, T. et al Nucleic Acids Res (1990) 18 : 3147-315
3; Glover, DMDNA Cloning; A Practical approachII
(1985) 143-190). These cell lines have been previously confirmed to be dexamethasone-inducible in myoblast formation and bmyf transcript expression as analyzed by Northern blot. These cell lines are used to analyze the turnover rate of bmyf transcripts. In addition, the bmyf protein produced and purified from these cell lines can be further characterized by, for example, bmf.
Used for analysis of yf protein phosphorylation status and localization.

Example 8 Antibody Polyclonal and monoclonal antibodies against bmyf protein can be obtained by using bmyf protein as an antigen. The methods and techniques utilized are those routinely used in the art for immunization, antibody selection, purification, hybridoma screening and immunoassays. Selection of such a combination of methods, as is routinely practiced by one of skill in the art, results in structural and functional properties of the bmyf protein as established in the previous examples, including immunoassay, staining, inactivation, affinity. Antibodies adapted to known techniques such as purification, epitope mapping, etc. are produced and purified. Typical antibody preparations are proteinaceous compositions containing either polyclonal or monoclonal antibodies, whether polyclonal or monoclonal, depending on the method used to obtain the antibodies. Antibodies specific for the bmyf protein are understood in the field of immunology to be antibodies that bind to bmyf. Since the bmyf protein has a homologous portion with other proteins, some antibodies specific to the bmyf protein may cross-react with other proteins to some extent. Antibodies that are tolerant of cross-reactivity for some purposes, but do not undergo measurable cross-reactivity with other proteins for other purposes are preferred.

Applicants are numbered 1-19, 101-118 and 154-1
Rabbit polyclonal antibodies were raised against three synthetic peptides containing the amino acid at position 69 (relative to the N-terminus). First, the region that seems to have antigenicity is PEPTIDESTR.
UCTURE ^TM Program (Jameson, BA & Wolf, H. (198
8) Comput. Appl. Biosci. 4 : 181-186; Pearson, WR &
Lippman, DJ (1988) PNAS USA 85 : 2444-2448; available from University of Wisconsin, Madison) and selected using a region with an antigenic index of at least 1,000. Antigen index can be calculated as hydrophilicity, surface accessibility, type of structure (eg, α,
β or β sheet) and Chou-Fas
man) is a compilation of peptide parameter information including characteristics. Furthermore, the selection of the antigenic peptide used to generate the antibody is (1) the length of the antigen region, (2) the presence of a strong β-fold structure in the predicted surface region, (3) the antigen region. The absence of cysteine in (4)
It was performed based on the absence of known homology with other proteins.

It has been found that an antigenic region of at least 15 residues is preferred. Because they are likely to present the antigenic region within a putative conformation in vivo. Also, the longer fragment potentially provides more epitopes recognized by the antibody,
This makes it easier to obtain specific antibodies. The presence of strong β-folding structures in the expected surface area is preferred. This is because the strong folded structure has often been found to retain its in vivo conformation also in vitro. The absence of cysteine in the antigenic region is preferred as it avoids reaction of the thiol groups with the reagents used to immobilize the antigenic peptide on the particles. The presence of cysteine in the region located adjacent to the antigenic region may be useful in anchoring the fragment to particles or supports. Cysteine can be inserted if it is not naturally present in the flanking region. To avoid such antibody cross-reactivity with other proteins, it is preferred that the antigenic region is heterologous to the other proteins. TFASTA program (Pearson, WR '
Lippman, DJ (1988) PNAS USA 85 : 2444-2448; available from University of Wisconsin, Madison, Wis.) Was used to translate the sequence of the antigenic region from the nucleotide sequence of EMBL registered in the Genebank database. All amino acid sequences obtained were compared. To avoid cross-reactivity with other proteins, the antigenic region used to raise the polyclonal antibody shares homology with all possible amino acid sequences translatable from the gene bank nucleotide sequence. Selected based on not being. When the above analysis was performed, the mouse myo D
Only (not bovine myo D) was obtained in the Genebank.

For each peptide selected, antisera were generated by the following method: (1) intramuscular administration of 50 μg of peptide mixed with complete Freund's adjuvant into rabbit muscle, (2) after 2 weeks, Intramuscularly administering 100 μg of the peptide mixed with incomplete Freund's adjuvant, (3) intramuscularly administering 100 μg of the peptide mixed with incomplete Freund's adjuvant weekly for 3 consecutive weeks, (4) ) Leave for 3 weeks, (5) Boost with 100 μg of peptide mixed with incomplete Freund's adjuvant, (6) Leave for 3 weeks,
And (7) mixed with incomplete Freund's adjuvant
Boost with 100 μg peptide. Serum was then collected and the reaction with bmyf and bovine myoD determined by immunoprecipitation.

It was found that each antiserum reacts with bmyf protein and cross-reacts with myoD protein in each antigenic peptide containing amino acids 1-19, 101-118 and 154-169. The polyclonal antibody precipitate can be further purified to reduce or eliminate antibodies that cross-react with myoD. This purification can be accomplished by previously known methods such as the antibody clearance method by affinity chromatography using myoD immobilized on a substrate. For example, the antibody preparation can be passed through a chromatography column in which the mammalian myoD protein or related proteins, or antigenic fragments thereof (including synthetic N-terminal fragments) are immobilized on a solid support or substrate, Results Antibodies reactive with myoD attach to the column. If desired, antibodies that are cross-reactive with other myogenic proteins can be removed as well. The eluate produced contains a high proportion of antibodies that are specific for the bmyf protein and do not react with bovine myoD.

[0081]

[Sequence listing] SEQ ID NO: 1 Sequence length: Nucleic acid 1931 Amino acid 255 Sequence type: Nucleic acid and amino acid Number of chains: Nucleic acid is double-stranded Topology: Linear sequence CCAGGCTCCG GTTTCTCCCC TATCTATCTC TCTGCTGTCC AGGCCCACCCCTTGCCTCTC 60 TGCAGACG ATG GAC ATG ATG GAC GGC TGC CAG TTC TCG CCC TCT GAG TAC 110 Met Asp Met Met Asp Gly Cys Gln Phe Ser Pro Ser Glu Tyr 5 10 TTC TAC GAC GGC TCC TGC ATC CCA TCC CCC GAC GGT GAG TTC GGG GAC 158 Phe Tyr Asp Gly Ser Cys Ile Pro Ser Pro Asp Gly Glu Phe Gly Asp 15 20 25 30 GAG TTT GAG CCG CGA GTG GCT GCT TTC GGG GCT CAC AAG GCA GAC CTG 206 Glu Phe Glu Pro Arg Val Ala Ala Phe Gly Ala His Lys Ala Asp Leu 35 40 45 CAA GGC TCA GAC GAG GAC GAG CAC GTG CGA GCA CCC ACG GGC CAC CAC 254 Gln Gly Ser Asp Glu Asp Glu His Val Arg Ala Pro Thr Gly His His 50 55 60 CAG GCC GGC CAC TGC CTC ATG TGG GCC TGC AAA GCA TGC AAA AGG AAG 302 Gln Ala Gly His Cys Leu Met Trp Ala Cys Lys Ala Cys Lys Arg Lys 65 70 75 TCC ACC ACC ATG GAT CGG CGG AA G GCG GCC ACC ATG CGC GAG CGG AGA 350 Ser Thr Thr Met Asp Arg Arg Lys Ala Ala Thr Met Arg Glu Arg Arg 80 85 90 CGC CTG AAG AAG GTC AAC CAG GCT TTC GAC ACG CTC AAG CGG TGC ACC 398 Arg Leu Lys Lys Val Asn Gln Ala Phe Asp Thr Leu Lys Arg Cys Thr 95 100 105 110 ACG ACC AAC CCT AAC CAG AGG CTG CCC AAG GTG GAG ATC CTC AGG AAT 446 Thr Thr Asn Pro Asn Gln Arg Leu Pro Lys Val Glu Ile Leu Arg Asn 115 120 125 GCC ATC CGC TAC ATT GAG AGT CTG CAG GAG CTG CTT AGG GAA CAG GTG 494 Ala Ile Arg Tyr Ile Glu Ser Leu Gln Glu Leu Leu Arg Glu Gln Val 130 135 140 GAA AAC TAC TAT AGC CTG CCG GGG CAG AGC TGC TCT GAG CCC ACC AGC 542 Glu Asn Tyr Tyr Ser Leu Pro Gly Gln Ser Cys Ser Glu Pro Thr Ser 145 150 155 CCC ACC TCA AGT TGC TCT GAT GGC ATG CCT GAA TGT AAC AGC CCT ATC 590 Pro Thr Ser Ser Cys Ser Asp Gly Met Pro Glu Cys Asn Ser Pro Ile 160 165 170 TGG TCC AGA AAG AGC AGC AGT TTT GAC AGC GTC TAC TGT CCT GAT GTA 638 Trp Ser Arg Lys Ser Ser Ser Phe Asp Ser Val Tyr Cys Pro Asp Val 175 180 185 190 CCA AAT GTA TAT GC C ACG GAT AAA AGC TCC TTA TCC AGC TTG GAT TGC 686 Pro Asn Val Tyr Ala Thr Asp Lys Ser Ser Leu Ser Ser Leu Asp Cys 195 200 205 TTA TCC AGC ATA GTG GAT CGG ATC ACC AAC TCA GAG CAA CCT GGA TTG 734 Leu Ser Ser Ile Val Asp Arg Ile Thr Asn Ser Glu Gln Pro Gly Leu 210 215 220 CCT CTC CAG GAT CCA GCC TCT CTC TCC CCA GTT GCC AGC ACC GAT TCT 782 Pro Leu Gln Asp Pro Ala Ser Leu Ser Pro Val Ala Ser Thr Asp Ser 225 230 235 CAA CCT GCA ACT CCA GGG GCC TCT AGT TCC AGG CTC ATC TAT CAT GTG 830 Gln Pro Ala Thr Pro Gly Ala Ser Ser Ser Arg Leu Ile Tyr His Val 240 245 250 CTA TGAACTAAAA ATCTAGATCA GTTCTGCCAG AAGGCCTATT ACACAGGAGG883 CAAAAACCCC AAAGCAAGAC AACCTGTAAA TAAACATTTC TTTTCTGTTG 943 TAAATTTGTA AATAGTTATC TTGCCACTTT ATAAGAAAGT GTATTTAAAA AGTCATTATT 1003 GCAATTTATA CTTTCTTTTT TTCTTCTTTT CCTTCATCTT TGCTTTAGAT ATATAGTTCC 1063 AATGATATTA TTTCTTATAG GGGCCGTTCA TCAAAGGTAG TTTGTTGCAA TGCTTAACTT 1123 ATATATTTTT ATAAATATTG CTTATCAAAA TATTACCTCT GATGTTTAGT GCTTTTTTTT 1183 CTTTAAAACA TTAGAA TAGA TGTAAATCAG TTATAGGGAG TTTTAAATAT ATTTAACTGT 1243 CTTGCTTTTC TTTAATCTTT TGATTTATAT TGTGTTAAGT AAAATATAAC AATACTGCCT 1303 AATGGTATAT ATTTTAACAT TTCTTATAAG AAATACATTT TTAATCTAAG CACAAAATAG 1363 TACTTTGTGG ATGATTTCAA GATATAAGAG ATTTTTGGAA ACTCCACCAT AAATAAAATT 1423 GTTTAAGTGA GGAAATATTC TGGTTTTATG ATTTTGTTAA AAGAACCCCC TAATGGAATT 1483 GGCAGTCACT GATATGGTGT CTTTAAGGTG GGGCTGAAAG TACTGAAACA GTGGGTTTTG 1543 AAGATAAATC TGAATAGCAT CTCGCGGACT CAGTTTAGGT CCTGTTGGTG TCACAGATGA 1603 AAATACCACA TTTCTACTTC AAGCATATTT CAAAGGTGTC TGCTAACTAA AATTTTTCTT 1663 TTATCTTAAT GTTGTACCAT ATAACATAAA GTTGTGTGTA TGTGGAGCAA AAGTAGAAGG 1723 TATTCAGGAC AATAACAGAT TTTTAAACAA AAAGAATGTA CAATATTACA TCATTATAAT 1783 ATCTATAGTA CATTAGAAAT CATGAGTTAA GGTGATATAT ATCTATTTTC CCAAAAATAC 1843 CTTGATACAT TAGTATATTA ATATTGTCAA TAAAATATTT AAAGGTAAAA AAAAAAAAAA 1903 AAAAAAAAAA AAAAAAAAAA AAAAAAAA 1931

SEQ ID NO: 2 Sequence length: 1931 Sequence type: Nucleic acid Number of strands: Single-stranded topology: Linear sequence CCAGGCTCCG GTTTCTCCCC TATCTATCTC TCTGCTGTCC AGGCCCACCC CTTGCCTCTC 60 TGCAGACG ATG GAC ATG ATG GAC GGC TGC CAG TTC TCG CCC TCT GAG TAC 110 TTC TAC GAC GGC TCC TGC ATC CCA TCC CCC GAC GGT GAG TTC GGG GAC 158 GAG TTT GAG CCG CGA GTG GCT GCT TTC GGG GCT CAC AAG GCA GAC CTG 206 CAA GGC TCA GAC GAG GAC GAG CAC GTG CGA GCA CCC ACG GGC CAC CAC 254 CAG GCC GGC CAC TGC CTC ATG TGG GCC TGC AAA GCA TGC AAA AGG AAG 302 TCC ACC ACC ATG GAT CGG CGG AAG GCG GCC ACC ATG CGC GAG CGG AGA 350 CGC CTG AAG AAG GTC AAC CAG GCT TTC GAC ACG CTC AAG CGG TGC ACC 398 ACG ACC AAC CCT AAC CAG AGG CTG CCC AAG GTG GAG ATC CTC AGG AAT 446 GCC ATC CGC TAC ATT GAG AGT CTG CAG GAG CTG CTT AGG GAA CAG GTG 494 GAA AAC TAC TAT AGC CTG CGC GGG CAGG TGC TCT GAG CCC ACC AGC 542 CCC ACC TCA AGT TGC TCT GAT GGC ATG CCT GAA TGT AAC AGC CCT ATC 590 TGG TCC AGA AAG AGC AGC AGT TTT GAC AGC GTC TAC TGT CCT GAT GTA 638 CCA AAT GTA TAT GCC ACG GAT AAA AGC TCC TTA TCC AGC TTG GAT TGC 686 TTA TCC AGC ATA GTG GAT CGG ATC ACC AAC TCA GAG CAA CCT GGA TTG 734 CCT CTC CAG GAT CCA GCC TCT TCC CCA GTT GCC AGC ACC GAT TCT 782 CAA CCT GCA ACT CCA GGG GCC TCT AGT TCC AGG CTC ATC TAT CAT GTG 830 CTA TGAACTAAAA ATCTAGATCA GTTCTGCCAG AAGGCCTATT ACACAGGAGG 883 AAGGAGGTAC CAAAAACCCC AAAGCAAGAC AACCTGTAAA TAAACATTTC TTTTCTGTTG 943 TAAATTTGTA AATAGTTATC TTGCCACTTT ATAAGAAAGT GTATTTAAAA AGTCATTATT 1003 GCAATTTATA CTTTCTTTTT TTCTTCTTTT CCTTCATCTT TGCTTTAGAT ATATAGTTCC 1063 AATGATATTA TTTCTTATAG GGGCCGTTCA TCAAAGGTAG TTTGTTGCAA TGCTTAACTT 1123 ATATATTTTT ATAAATATTG CTTATCAAAA TATTACCTCT GATGTTTAGT GCTTTTTTTT 1183 CTTTAAAACA TTAGAATAGA TGTAAATCAG TTATAGGGAG TTTTAAATAT ATTTAACTGT 1243 CTTGCTTTTC TTTAATCTTT TGATTTATAT TGTGTTAAGT AAAATATAAC AATACTGCCT 1303 AATGGTATAT ATTTTAACAT TTCTTATAAG AAATACATTT TTAATCTAAG CACAAAATAG 1363 TACTTTGTGG ATGATTTCAA GATATAAGAG ATTTTTGGAA ACTCCACCAT AAATAAAATT 1423 GTTTAAGTGA GGAAATATTC TGGTTTTATG ATTTTGTTAA AAGAACCCCC TAATGGAATT 1483 GGCAGTCACT GATATGGTGT CTTTAAGGTG GGGCTGAAAG TACTGAAACA GTGGGTTTTG 1543 AAGATAAATC TGAATAGCAT CTCGCGGACT CAGTTTAGGT CCTGTTGGTG TCACAGATGA 1603 AAATACCACA TTTCTACTTC AAGCATATTT CAAAGGTGTC TGCTAACTAA AATTTTTCTT 1663 TTATCTTAAT GTTGTACCAT ATAACATAAA GTTGTGTGTA TGTGGAGCAA AAGTAGAAGG 1723 TATTCAGGAC AATAACAGAT TTTTAAACAA AAAGAATGTA CAATATTACA TCATTATAAT 1783 ATCTATAGTA CATTAGAAAT CATGAGTTAA GGTGATATAT ATCTATTTTC CCAAAAATAC 1843 CTTGATACAT TAGTATATTA ATATTGTCAA TAAAATATTT AAAGGTAAAA AAAAAAAAAA 1903 AAAAAAAAAA AAAAAAAAAA AAAAAAAA 1931

SEQ ID NO: 3 Sequence length: 255 Sequence type: Amino acid Topology: Linear sequence Met Asp Met Met Asp Gly Cys Gln Phe Ser Pro Ser Glu Tyr Phe Tyr 5 10 15 Asp Gly Ser Cys Ile Pro Ser Pro Asp Gly Glu Phe Gly Asp Glu Phe 20 25 30 Glu Pro Arg Val Ala Ala Phe Gly Ala His Lys Ala Asp Leu Gln Gly 35 40 45 Ser Asp Glu Asp Glu His Val Arg Ala Pro Thr Gly His His Gln Ala 50 55 60 Gly His Cys Leu Met Trp Ala Cys Lys Ala Cys Lys Arg Lys Ser Thr 65 70 75 80 Thr Met Asp Arg Arg Lys Ala Ala Thr Met Arg Glu Arg Arg Arg Leu 85 90 95 Lys Lys Val Asn Gln Ala Phe Asp Thr Leu Lys Arg Cys Thr Thr Thr 100 105 110 Asn Pro Asn Gln Arg Leu Pro Lys Val Glu Ile Leu Arg Asn Ala Ile 115 120 125 Arg Tyr Ile Glu Ser Leu Gln Glu Leu Leu Arg Glu Gln Val Glu Asn 130 135 140 Tyr Tyr Ser Leu Pro Gly Gln Ser Cys Ser Glu Pro Thr Ser Pro Thr 145 150 155 160 Ser Ser Cys Ser Asp Gly Met Pro Glu Cys Asn Ser Pro Ile Trp Ser 165 170 175 Arg Lys Ser Ser Ser Phe Asp Ser Val Tyr Cys Pro Asp Val Pro Asn 180 185 190 Val Tyr Ala Thr Asp Lys Ser Ser Leu Ser Ser Leu Asp Cys Leu Ser 195 200 205 Ser Ile Val Asp Arg Ile Thr Asn Ser Glu Gln Pro Gly Leu Pro Leu 210 215 220 Gln Asp Pro Ala Ser Leu Ser Pro Val Ala Ser Thr Asp Ser Gln Pro 225 230 235 240 Ala Thr Pro Gly Ala Ser Ser Ser Arg Leu Ile Tyr His Val Leu 245 250 255

SEQ ID NO: 4 Sequence Length: 385 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence TTGAGATTGG TAGAGAAGTC TTGGGTTGCT ATAGTGACCT TCACCCTTAT TAAGACATTT 60 AAATTCAAAG CTAGAATTTA CCCATCCATCTGTG CTTTTCTCTT TAGCCTCATTTTTTGTTAGCATACATTTTGTTAGCATAATTTTTTGT TTTTAAAAAA 180 TTTTAGTTCA AACTTTGAAT ATCCATCTTC CAAATCTAAT GCATTTGTAA ATTAGCTATA 240 ACATGGCATT TCCTTAAAGA GCCAAGCTAT CAAGGCATTT CTCCAGCAGA CTCCTCCAGC 300 CACTCCCTTA GAGTCAGCAGGCTGTGTCTTGATCGCCAGCAGTCATCGACAGCGCTGA CTGACTGAGCT

SEQ ID NO: 5 Sequence Length: 814 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence GAGCTCAAGC CCTCCAGAAG CGATGCCTAG TTTCCAATTC TGAGTGACCT GGGGCAAATG 60 ACTTACTCCC CGAGCTTGGC TACCTTCTTT TAATAATGGGTCACAG GTCCACGTAGA AGCTTCCCAA 180 GAGAAAACTC TTCTAATTGT TTGCTTACAA ATATCCATGC TTAACAACTT TGTGCTTTGT 240 GGAAAGAAAT CGCCTAAAGA CAAGCCAGAA GCATAACTTC TATCTTCCAC TTAAACTTGA 300 AGGAGCAAAT TTATTTGTGC CCGAATCTTC AATTTGAACA CAGGTCTGAT TTGCTTTGAC 360 CAGTGTGTGA CAGTCTGGAC TACAGACCCA AAGAAAGTGC TACTATAGCT GCTGACCAGA 420 ACCTGTGGTC CCTGGCTGAG AATTTCTGGA TGAGAATATC TACCCACTAC TGTGCTTCTG 480 AGGTGGCAAC GTGCACACTT TGGAATGTTT CCACTCCCCT GGATGGGTCC TTTAGGAACT 540 CAATTCTTTT ATCTGGTGTA GCCTGGGTAC CTATTCATTT CAAATGCATC TACTTGTTAA 600 CAGTTCTAAA AATAAGGTGC AGATGAATTC CCACTGCCCA ATTAAGCTGT CCATTGGTAT 660 GAAAAGGAGC CAGATTTTCA AGGGATGGAT GGTGATGGTG GTGGTGGAGG TAGTGTCTTT 720 GTCTTGGAGA TGACAG AGCT TGGGAAAGGG GACAGCGGCA GGCACGTTTG TAGCAAACTT 780 TGGTGACTGA CATTCTCCTA CCCCTGATTT TGCT 814

SEQ ID NO: 6 Sequence length: 946 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear sequence GGGAAAAGGG AAGCAAGCCC TGGAAGAGTT CATCATCTAA ACCTGGTTTC TTAGAATACA 60 AATCTTAGAG ACGCAGTCCA CAGAGAGCAG CAGCAGGACCATCAGAGCACACAGCAGAGAGCAGCAGAGAGCAGCAGCAGCAGCAAGCAGCAGCAGCAGCAGCAGAGCAGCAGCAGCAGAGCAGCAGCAGCAGAGCAGCAGCAGCAGAGCAGCAGCAGCAGAGCAGCAGAGAGCAGCAGAGCAGCAGAGCAGCAGAGAGCAGCAGAGCAGCAGAGCAGCAGAGAGCAGCAGAGCAGCAGAGAGCAGCAGAGAGCAGCAGAGCAGCAGAGCAGCAGAGAGCAGCAGAGCAGCAGAGAGCAGCAGAGCAGCAGAGAGCAGCAGGACAGAGCAGCAGAGCAGCAGGAGCAGCAGCAGGACGAGACCAGAGACCAGAGAGACCAGAGACAGCAGACGAGACCAGACGAGACCACHAG. CCTGGTCACC 180 TGGCCCTCCT GTGCCCACAC CCTTCAGATG AGCAGAGAGA AAACTCAGAG CAAGCTGTTT 240 TTGGAGAAAA GGAACCTGCC AGGTTACCCC CAATAACATA CAGCCTACAT AATTGCAATC 300 TGATGAGAAA CAAAACAGAT TAAAAAAAAA AAATCTAGAG CCACTTCATT ATTGAAAGTG 360 CCACTTGAAG TTTTTAGCAA CTGTTATTTG GCCTAATGGC TGAAAAAATT CCTGACACAA 420 CTAAAGTTCA CGAAAAAGAG AAATACCTGA AAATAAAGTT GGTCCCTCAT GTCTCATATT 480 GGGAAGACGG GTCCGCTTTT GGTGTCAACT GAAATGGGTA GCTGTATTTA CCGATTTTCT 540 TTTTAAATGC GGAGAGCAAT TGTACAAGTT GGATTTCCTT CGTTGCTTTT GTCTGTGACA 600 CCCTCTAAGT GTCCCTTGGT TTAGGTAATC TAAAGAGTGC AAGTAAGCGG ATTGGGGAGC 660 CATCACCTCC GCCCCAGCAG AAACGCAGAA ACCATTAGCG TCAAAAGGGG CAGTAAACAG 720 CGACTGTCTC TAGAGA AAAG GCGGAGGTTT GCCCAGACAG CACTGGCGGG GGTTGCGGTG 780 GGATATGCTA ATAGTGCGGG GCCACAGGGG CGGCCTCCCT CCCCCAAGAA TATATAAAGA 840 GCCCCAGCCC CAGCGCGCAG ACCCCAGGCC GCCACGTCTGCCCTCTCTTTAA TTACCAGAGA 900 CACCGACCAG GGA

SEQ ID NO: 7 Sequence length: 569 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear sequence CCAGGCTCCG GTTTCTCCCC TATCTATCTC TCTGCTGTCC AGGCCCACCC CTTGCCTCTC 60 TGCAGACGAT GGACATG ATG GAC GGC TGC CAG TTC TCG CCC TCT GCT TAC 110 TTC TAC GAC GGC TCC TGC ATC CCA TCC CCC GAC GGT GAG TTC GGG GAC 158 GAG TTT GAG CCG CGA GTG GCT GCT TTC GGG GCT CAC AAG GCA GAC CTG 206 CAA GGC TCA GAC GAG GAC GAG CAC GTG CGA GCA CCC ACG G CAC CAC 254 CAG GCC GGC CAC TGC CTC ATG TGG GCC TGC AAA GCA TGC AAA AGG AAG 302 TCC ACC ACC ATG GAT CGG CGG AAG GCG GCC ACC ATG CGC GAG CGG AGA 350 CGC CTG AAG AAG GTC AAC CAG GCT TTC GAC ACG CTC AAG CGG TGC ACC 398 ACG ACC AAC CCT AAC CAG AGG CTG CCC AAG GTG GAG ATC CTC AGG AAT 446 GCC ATC CGC TAC ATT GAG AGT CTG CAG GAG CTG CTT AGG GAA CAG GTG 494 GAA AAC TAC TAT AGC CTG CCG GGG CACT AGC GAG CCC ACC AGC 542 CCC ACC TCA AGT TGC TCT GAT GGC ATG 569

SEQ ID NO: 8 Sequence Length: 361 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence GTAAGAGATG GCTCTGTACC TGCTAGGACC TTCCAACTTT TACAAAAATC CTTACATCTC 60 ATTTAGACCGAGGTGTAGCACA CCAGATATTC AGTGGTTACT GCGGGTAGGTAGGAGGTAGCATGAGCATGAGCATGAGCATG AGACAGATGC 180 CAGCACACAG ACAGCTGTTG AACAGGATTT TGTCCAGACT TCCTACCTAC TCTAGGTGCA 240 CACTGAAGGA GCAGGTTGTC ACTTGAGATA GCTGGCTGTG AATGATCAGT CAGATTTCTC 300 CATTACTCAG TTATTCAGTG TTATACTACC AGTGGACCTT GACATACCTA ACATTA

SEQ ID NO: 9 Sequence Length: 258 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence ATTTGCATGT AACCAGTGTA CACTGCTGCT GAA
TTCCACT CTCTTTCTTTC TGCTGCCTCT 60 CTCCTCTTTCT CCAAGCACAG AGATTGACCT CAG
TGCCCTT GCAATTGGTG AGGCTAGCCC 120 TTCCTAAATC AAGGGCAATAA AGGTGATTGA GAG
TGGTCAG TTTATGGAGC TGGTGGGGCAA 180 GCACTGCCCTC ACCCGAGATT GGAGCCACTT TTG
CCAAGAC CTGAAAAACAA ACGCCTTCTT 240 CTGTGTCTTT TATTATAG
258

SEQ ID NO: 10 Sequence Length: 75 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence CCT GAA TGT AAC AGC CCT ATC TGG TCC
AGA AAG AGC AGC AGT TTT GAC 48 AGC GTC TAC TGT CCT GAT GTA CCA AAT
75

SEQ ID NO: 11 Sequence length: 393 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear sequence GGGTAAGACC AATGACTTCA AGAGAGTTAA GACCAGGTTC AACTTCAGAT TAGCCTATCA 60 AGTAGAGTCT GGTTCTTCCA TGCAGAGTACTG GGAAGATCACACAGTCATCACACA GATGGTATTA 120 TCCATGATAT 180 ACACACAGAC GTGAGCTTTT GCTTGAAATA TTACCCAGGT GCTTCTCCAC TCCCCAACTT 240 CATCCCCATG AATTGCTAGA TATTTGTTGC AAATTTCTAC CAGGCTTTCT GTGACCACCT 300 GACCTTTGGG TTTCAAAGGT GGTGACCTTC AGTTTAAGCG GCAGCTTAGCTTT

SEQ ID NO: 12 Sequence length: 1245 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear sequence GTA TAT GCC ACG GAT AAA AGC TCC TTA TCC AGC TTG GAT TGC TTA TCC 48 AGC ATA GTG GAT CGG ATC ACC AAC TCA GAG CAA CCT GGA TTG CCT CTC 96 CAG GAT CCA GCC TCT CTC TCC CCA GTT GCC AGC ACC GAT TCT CAA CCT 144 GCA ACT CCA GGG GCC TCT AGT TCC AGG CTC ATC TAT CAT GTG CAA TGA 192TA TAGATCAGTT CTGCCAGAAG GCCTATTACA CAGGAGGAAG GAGGTACCAA 252 AAACCCCAAA GCAAGACAAC CTGTAAATAA ACATTTCTTT TCTGTTGTAA ATTTGTAAAT 312 AGTTATCTTG CCACTTTATA AGAAAGTGTA TTTAAAAAGT CATTATTGCA ATTTATACTT 372 TCTTTTTTTC TTCTTTTCCT TCATCTTTGC TTTAGATATA TAGTTCCAAT GATATTATTT 432 CTTATAGGGG CCGTTCATCA AAGGTAGTTT GTTGCAATGC TTAACTTATA TATTTTTATA 492 AATATTGCTT ATCAAAATAT TACCTCTGAT GTTTAGTGCT TTTTTTTCTT TAAAACATTA 552 GAATAGATGT AAATCAGTTA TAGGGAGTTT TAAATATATT TAACTGTCTT GCTTTTCTTT 612 AATCTTTTGA TTTATATTGT GTTAAGTAAA ATATAACAAT ACTGCCTAAT GGTATATATT 672 TTAACATTTC TTATAAGAA A TACATTTTTA ATCTAAGCAC AAAATAGTAC TTTGTGGATG 732 ATTTCAAGAT ATAAGAGATT TTTGGAAACT CCACCATAAA TAAAATTGTT TAAGTGAGGA 792 AATATTCTGG TTTTATGATT TTGTTAAAAG AACCCCCTAA TGGAATTGGC AGTCACTGAT 852 ATGGTGTCTT TAAGGTGGGG CTGAAAGTAC TGAAACAGTG GGTTTTGAAG ATAAATCTGA 912 ATAGCATCTC GCGGACTCAG TTTAGGTCCT GTTGGTGTCA CAGATGAAAA TACCACATTT 972 CTACTTCAAG CATATTTCAA AGGTGTCTGC TAACTAAAAT TTTTCTTTTA TCTTAATGTT 1032 GTACCATATA ACATAAAGTT GTGTGTATGT GGAGCAAAAG TAGAAGGTAT TCAGGACAAT 1092 AACAGATTTT TAAACAAAAA GAATGTACAA TATTACATCA TTATAATATC TATAGTACAT 1152 TAGAAATCAT GAGTTAAGGT GATATATATC TATTTTCCCA AAAATACCTT GATACATTAG 1212 TATATTAATA TTGTCAATAA AATATTTAAA GGT 1245

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C12P 21/08 8214-4B // (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91) 8828-4B C12N 15/00 C (72) Inventor Robert Day. I've Ally United States Giosia 30677 Watkinsville, Jackson Street 54 (72) Inventor Jurie A .. Morris United States Giosia 30677 Watkinsville, Jackson Street 54 (72) Inventor Theodor G. Clark United States of America Giosia 30605 Assens, Morton Avenyeu 190 (72) Inventor Chiarz K. Smith United States Indiana 46140 Greenfield, Arrowhead Drive 5550 (72) Inventor William F. Heath, The Uninia United States Indiana 46250 Indianapolis, San Diego Drive 9502

Claims (23)

[Claims] 1. A cloned DNA containing a nucleotide sequence encoding a bmyf active protein. 2. The DNA of claim 1, wherein the sequence encodes the bmyf protein. 3. The cloned DNA is cDNA.
The DNA according to claim 1. 4. The DNA according to claim 1, wherein the cloned DNA is a cDNA having a nucleotide sequence encoding the bmyf protein shown in SEQ ID NO: 1. 5. The DNA according to claim 1, wherein the cloned DNA is genomic DNA. 6. The DNA of claim 5, wherein the cloned DNA comprises one or more sequences selected from the group of SEQ ID NOs: 3-11. 7. A cultured cell transformed with the cloned DNA of claim 1. 8. A D containing a promoter of bmyf gene
NA. 9. The DNA according to claim 8, wherein the promoter comprises one or more sequences selected from the sequence group of SEQ ID NOS: 3, 4 and 5. 10. The DNA of claim 8, wherein the promoter comprises a bmyf active promoter. 11. The DNA according to claim 8, which further comprises a coding region under the control of the promoter. 12. The DNA according to claim 11, wherein the coding region is a heterologous coding region. 13. The DNA according to claim 11, wherein the coding region encodes a reporter gene. 14. The DNA according to claim 11, wherein the coding region encodes a bmyf active protein. 15. The DNA according to claim 11, wherein the coding region encodes a bmyf protein. 16. A cultured cell transformed with the promoter and coding region of claim 11. 17. A purified protein having an amino acid sequence containing the amino acid sequence of bmyf active protein. 18. The protein according to claim 17, wherein the amino acid sequence comprises the amino acid sequence of bmyf protein. 19. A proteinaceous composition comprising a polyclonal or monoclonal antibody specific for bmyf protein. 20. A method according to claim 19, which comprises a monoclonal antibody.
The antibody according to. 21. Bmyf amino acids 1 to 19 of SEQ ID NO: 3
Including a polyclonal antibody against a peptide having
The antibody according to claim 19. 22. Bmyf amino acids 101 to SEQ ID NO: 3
20. The antibody of claim 19, which comprises a polyclonal antibody against a peptide having 118. 23. The bmyf amino acids 154 to SEQ ID NO: 3
20. The antibody of claim 19, comprising a polyclonal antibody against a peptide having 169.